Stem Cell Banking: Concepts and Protocols

Methods in
Molecular Biology 1590
Jeremy M. Crook
Tenneille E. Ludwig Editors
Stem Cell
Banking
Concepts and Protocols
METHODS IN MOLECULAR BIOLOGY
Series Editor
John M. Walker
School of Life and Medical Sciences
University of Hertfordshire
Hatfield, Hertfordshire, AL10 9AB, UK
For further volumes:

http://www.springer.com/series/7651
Stem Cell Banking
Concepts and Protocols
Edited by
Jeremy M. Crook
ARC Centre of Excellence for Electromaterials Science, Intelligent Polymer Research Institute, AIIM Facility,
Innovation Campus, University of Wollongong, Fairy Meadow, NSW, Australia
Illawarra Health and Medical Research Institute, University of Wollongong, Wollongong, NSW, Australia
Department of Surgery, St Vincent’s Hospital, The University of Melbourne, Fitzroy, VIC, Australia
Tenneille E. Ludwig
WiCell Research Institute, Madison, WI, USA
Editors
Jeremy M. Crook Tenneille E. Ludwig
ARC Centre of Excellence for Electromaterials WiCell Research Institute
Science Madison, WI, USA
Intelligent Polymer Research Institute
AIIM Facility, Innovation Campus
University of Wollongong
Fairy Meadow, NSW, Australia
Illawarra Health and Medical Research Institute
University of Wollongong
Wollongong, NSW, Australia
Department of Surgery
St Vincent’s Hospital
The University of Melbourne
Fitzroy, VIC, Australia
ISSN 1064-3745 ISSN 1940-6029 (electronic)

Methods in Molecular Biology
ISBN 978-1-4939-6919-7 ISBN 978-1-4939-6921-0 (eBook)
DOI 10.1007/978-1-4939-6921-0
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Preface
Stem cell banking has a critical role to play for supporting high quality research and transcend-
ing the clinical potential of stem cells to actual medicine. Ideally, this is achieved by operating
within a regulatory framework of good laboratory practice (GLP) or good manufacturing
practice (GMP) for standardized, optimized, and controlled cell line production, storage, and
distribution. Among other benefits, creating repositories of quality “seed stock” is a most
immediate way to circumvent the problems associated with extended cell culture, including
susceptibility to genetic and phenotypic drift during propagation, loss of cells due to cross-
contamination with microorganisms or other cell lines, and stem cell differentiation.
In recognizing the need for modern banking systems, major developed nations includ-
ing the US, UK, and Japan have invested significantly in stem cell banking to prepare for
the next major phase in researching and commercializing stem cells and producing clinical
treatments.
Importantly, stem cell banking need not entail setting up large and expensive stand-
alone facilities that operate on a national or international scale, but can involve smaller ini-
tiatives to support the activities of individual universities, research institutes, or laboratories.
Whatever the scale, a bank should align with global “best practice” for handling stem cells,
ideally endorsed by leading stem cell organizations, networks, and consortia around the
world. Moreover, a bank should ensure the management and distribution of cell lines in the
most efficient and cost-effective way. For example, the succession of commercial and clinical
aspirations could be facilitated by having low-cost quality-controlled GLP cells for research
that are also available as more expensive clinical-grade GMP lines. In addition, research and
clinical-grade variants of the same cell lines/banks will provide consistency between labora-
tory and clinical activities for more predictable and better translational application.
Given the recent upsurge in stem cell research and development (R&D), including
technological breakthroughs in creating new types of stem cells such as induced pluripotent
stem cells (iPSCs), as well as clinical trials of human stem cell-based therapies, the publica-
tion of this book on Stem Cell Banking is timely.
This volume brings together contributions from experts in the field to guide stem cell
banking, and in turn champion quality stem cell R&D and facilitate the translation of stem
cells to clinical practice. The book covers concepts and protocols relating to the banking of
both pluripotent and somatic stem cells, from the ethical procurement of tissues and cells
for the provision of “seed stock,” standardized methods for deriving hESCs and iPSCs,
isolating mesenchymal stem cells, cell culture and cryopreservation, in addition to quality
assurance (including cell line characterization) and information management.
As a volume in the highly successful Methods in Molecular Biology™ series, it aims to
contribute to the development of competence in the subject by providing advice that is crucial
to establishing a bona fide stem cell bank. By proffering Stem Cell Banking, we hope to
strengthen and maximize the use of existing and future stem cell resources. Finally, the volume
should serve as a valuable resource for established stem cell scientists and those new to the field.
Wollongong, NSW, Australia Jeremy M. Crook
v
Contents
Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix
PART I GENERIC THEMES IN STEM CELL BANKING

1 Stem Cell Banking: A Global View . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Glyn Stacey
2 Quality Assurance in Stem Cell Banking: Emphasis on Embryonic
and Induced Pluripotent Stem Cell Banking . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Therése Kallur, Pontus Blomberg, Sonya Stenfelt, Kristian Tryggvason,
and Outi Hovatta
3 Acquisition and Reception of Primary Tissues, Cells,
or Other Biological Specimens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Lyn E. Healy
4 Information Management . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
Alberto Labarga, Izaskun Beloqui, and Angel G. Martin
5 Cryopreservation: Vitrification and Controlled Rate Cooling. . . . . . . . . . . . . . 41
Charles J. Hunt
6 Quality Assured Characterization of Stem Cells for Safety in Banking
for Clinical Application . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79
Kevin W. Bruce, John D.M. Campbell, and Paul De Sousa
7 Ethics and Governance of Stem Cell Banks . . . . . . . . . . . . . . . . . . . . . . . . . . . 99
Donald Chalmers, Peter Rathjen, Joy Rathjen, and Dianne Nicol
PART II PROTOCOLS FOR PLURIPOTENT STEM CELL BANKING

8 Derivation of Human Embryonic Stem Cells. . . . . . . . . . . . . . . . . . . . . . . . . . 115
Jeremy M. Crook, Lucy Kravets, Teija Peura, and Meri T. Firpo
9 Derivation of Human-Induced Pluripotent Stem Cells in Chemically
Defined Medium. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131
Guokai Chen and Mahendra Rao
10 Culture, Adaptation, and Expansion of Pluripotent Stem Cells . . . . . . . . . . . . 139
Jennifer L. Brehm and Tenneille E. Ludwig
11 Cryobanking Pluripotent Stem Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151
Jeremy M. Crook, Eva Tomaskovic-Crook, and Tenneille E. Ludwig
12 Genome Editing in Human Pluripotent Stem Cells . . . . . . . . . . . . . . . . . . . . . 165
Jared Carlson-Stevermer and Krishanu Saha
vii
viii Contents
PART III PROTOCOLS FOR MESENCHYMAL STEM CELL BANKING

13 Isolation, Culture, and Expansion of Mesenchymal Stem Cells . . . . . . . . . . . . 177
Izaskun Ferrin, Izaskun Beloqui, Lorea Zabaleta, Juan M. Salcedo,
Cesar Trigueros, and Angel G. Martin
14 Cryobanking Mesenchymal Stem Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 191
Andrés Pavón, Izaskun Beloqui, Juan M. Salcedo, and Angel G. Martin
PART IV PROTOCOLS FOR HUMAN NEURAL STEM CELL BANKING

15 Culturing and Cryobanking Human Neural Stem Cells . . . . . . . . . . . . . . . . . . 199
Jeremy M. Crook and Eva Tomaskovic-Crook
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 207
Contributors
IZASKUN BELOQUI • StemTek Therapeutics, Derio, Spain

PONTUS BLOMBERG • Vecura, Karolinska University Hospital, Stockholm, Sweden
JENNIFER L. BREHM • WiCell Research Institute, Madison, WI, USA
KEVIN W. BRUCE • Censo Biotechnologies Ltd and Roslin Cell Sciences Ltd, Midlothian, UK
JOHN D.M. CAMPBELL • Scottish Blood Transfusion Service, Edinburgh, UK
JARED CARLSON-STEVERMER • Department of Biomedical Engineering, University of
Wisconsin-Madison, Madison, WI, USA; Wisconsin Institute for Discovery, University
of Wisconsin-Madison, Madison, WI, USA
DONALD CHALMERS • Centre for Law and Genetics, Faculty of Law, University of
Tasmania, Hobart, TAS, Australia
GUOKAI CHEN • Faculty of Health Sciences, University of Macau, Taipa, Macau, China;
Center for Molecular Medicine, National Heart, Lung and Blood Institute, Bethesda,
MD, USA
JEREMY M. CROOK • ARC Centre of Excellence for Electromaterials Science, Intelligent
Polymer Research Institute, AIIM Facility, Innovation Campus, University of
Wollongong, Fairy Meadow, NSW, Australia; Illawarra Health and Medical Research
Institute, University of Wollongong, Wollongong, NSW, Australia; Department of
Surgery, St Vincent’s Hospital, The University of Melbourne, Fitzroy, VIC, Australia
IZASKUN FERRIN • StemTek Therapeutics, Derio, Spain
MERI T. FIRPO • Division of Endocrinology and Stem Cell Institute, Department
of Medicine, McGuire Translational Research Facility, University of Minnesota,
Minneapolis, MN, USA
LYN E. HEALY • The Francis Crick Institute, London, UK
OUTI HOVATTA • CLINTEC, Karolinska Institute, Flemingsberg, Sweden
CHARLES J. HUNT • UK Stem Cell Bank, National Institute for Biological Standards and
Control, Hertfordshire, UK
THERÉSE KALLUR • BioLamina, Stockholm, Sweden
LUCY KRAVETS • Centre for Blood Cell Therapies, Peter MacCallum Cancer Centre, East
Melbourne, Australia
ALBERTO LABARGA • Department of Computer Science and Artificial Intelligence,
University of Granada, Gardana, Spain
TENNEILLE E. LUDWIG • WiCell Research Institute, Madison, WI, USA
ANGEL G. MARTIN • StemTek Therapeutics, Derio, Spain
DIANNE NICOL • Centre for Law and Genetics, Faculty of Law, University of Tasmania,
Hobart, TAS, Australia
ANDRÉS PAVÓN • StemTek Therapeutics, Derio, Spain
TEIJA PEURA • Genea Biomedx, Sydney, NSW, Australia
MAHENDRA RAO • New York Stem Cell Foundation Research Institute, New York, NY,
USA; Q Therapeutics, Salt Lake City, UT, USA; Wake Forest Institute for Regenerative
Medicine, Wake Forest University, Winston-Salem, NC, USA
ix
x Contributors
JOY RATHJEN • School of Medicine, University of Tasmania, Hobart, TAS, Australia

PETER RATHJEN • The Menzies Institute of Medical Research, University of Tasmania,
Hobart, TAS, Australia
KRISHANU SAHA • Department of Biomedical Engineering, University of
Wisconsin-Madison, Madison, WI, USA; Wisconsin Institute for Discovery,
University of Wisconsin-Madison, Madison, WI, USA
JUAN M. SALCEDO • StemTek Therapeutics, Derio, Spain
PAUL DE SOUSA • Roslin Cell Sciences Ltd., Midlothian, UK; Censo Biotechnologies Ltd.,
Midlothian, UK; Centre for Clinical Brain Sciences, University of Edinburgh,
Edinburgh, UK
GLYN STACEY • UK Stem Cell Bank, National Institute for Biological Standards and
Control, Hertfordshire, UK
SONYA STENFELT • Department of Neuroscience, Uppsala University, Uppsala, Sweden
EVA TOMASKOVIC-CROOK • AIIM Facility, ARC Centre of Excellence for Electromaterials
Science, Intelligent Polymer Research Institute, University of Wollongong, Fairy Meadow,
NSW, Australia; Illawarra Health and Medical Research Institute, University of
Wollongong, Wollongong, NSW, Australia
CESAR TRIGUEROS • StemTek Therapeutics, Derio, Spain
KRISTIAN TRYGGVASON • BioLamina, Stockholm, Sweden
LOREA ZABALETA • StemTek Therapeutics, Derio, Spain
Part I
Generic Themes in Stem Cell Banking

Chapter 1
Stem Cell Banking: A Global View

Glyn Stacey
Abstract
Stem cell banking has been a topic of discussion and debate for more than a decade since the first public
services to supply human embryonic stem cells (hESCs) were established in the USA and the UK. This
topic has received a recent revival with numerous ambitious programmes announced to deliver large col-
lections of human induced pluripotency cell (hiPSC) lines. This chapter will provide a brief overview chart-
ing the development of stem cell banks, their value, and their likely role in the future.
Key words Pluripotent stem cell banks, Human embryonic stem cells, Induced pluripotent stem cells,
Rationale, History, Challenges
1 The Rationale for Stem Cell Banks
In all research using cell lines the scientific quality of the source
cells is crucial. The exchange of cell lines between researchers is
part of the traditional scientific currency securing inter-laboratory
collaboration. However, all too often cells exchanged in this way
have become genetically altered during culture passage, switched
or cross-contaminated with another cell line, or contaminated with
mycoplasma, which often leads to permanent adverse genetic and/
or phenotypic change [1]. The consequences for research per-
formed with the wrong, altered, and/or mixed cell cultures are
clearly serious for the validity of any resulting published work and
can lead to retraction of publications. While the originators of cell
lines may pay special attention to supply suitable cells to collabora-
tors, it has been shown in cancer cell lines that a significant propor-
tion of cells volunteered for deposit in public collections are no
longer the original cell line (i.e., switched with another cell line)
and more seriously, numerous examples were provided by the orig-
inators of the lines themselves [2]. Already, examples of cross-con-
taminated and mycoplasma contaminated cells have been identified
among the hESCs available for research and in other cases the cell
line has become overgrown by a chromosomally abnormal clone.
Jeremy M. Crook and Tenneille E. Ludwig (eds.), Stem Cell Banking: Concepts and Protocols, Methods in Molecular Biology, vol. 1590,
DOI 10.1007/978-1-4939-6921-0_1, © Springer Science+Business Media LLC 2017
3
4 Glyn Stacey
In recent work to establish the European bank of iPSC lines (www.

ebisc.org/), early submissions were exposed to comprise 14% hiP-
SCs lines that were not from the correct donor (J Holder, personal
communication). This situation requires all researchers to take
responsibility for assuring the characteristics of the cells they use,
and is a driver for the existence of resource centers focused on the
establishment of quality controlled seed stocks that will provide for
long-term supply of stem cell lines for research.
Centers dedicated to the long-term supply of stem cell lines
provide the opportunity for researchers to secure a number of addi-
tional benefits. These include safe backup stocks, access to advice,
and training in the culture and preservation of a range of different
cell lines and in some cases such as the UK Stem Cell Bank (SCB) a
patent depositary. A further and significant benefit from stem cell
banking is the assurance of good practice. A network of stem cell
banking centers and individuals and organizations committed to
supporting formalized stem cell banking called the International
Stem Cell Banking Initiative (ISCBI) has produced a consensus on
principles of best practice in the procurement, banking testing, and
distribution of hESC lines for research [3]. ISCBI has coordinated
international opinion on requirements for high-quality hiPSC
banking (2012 meeting report: http://www.stem-cell-forum.net/
initiatives/international-stem-cell-banking-initiative/) and com-
pleted a consensus on development of seed stock of pluripotent
stem cell lines for clinical application [4].
Unfortunately, the technical challenge and cost of banking
cells has meant that operating such collections, of even the most
readily expanded cell lines, means that such resources do little
more than recover costs and require ongoing institutional subsidy
to sustain them (for example, banking a research-grade and clinical-
grade cell line costs approximately £60 thousand and £1 million
respectively when all staff, facilities, safety testing, and overheads
costs are accounted for). This is demonstrated by the very limited
number of banks engaged in the supply of human pluripotent stem
cell (hPSC) lines internationally at any significant level. Significant
improvements including advanced technologies for increased effi-
ciencies in cell culture will be required to make self-supporting
stem cell banking a reality.
2 History of Banking Human Pluripotent Stem Cell Lines
The discovery that hESCs could be generated from human blasto-

cysts in 1998 rightly caused great excitement. As pluripotent cells
can give rise to cells of the three germ layers required to form the
tissues of the human body, they gave hope to providing broad
Stem Cell Banking: A Global View 5
ranging restorative therapy to replace damaged or diseased tissue

[5]. This potential was immediately recognized by the US National
Institutes of Health (NIH) who funded the providers of lines to
supply them worldwide. Following UK legislation in 2001 to enable
the generation and sharing of hESC lines between researchers, the
Medical Research Council (MRC) coordinated a project to estab-
lish regulatory oversight and a national bank to ensure that stocks
of hESCs were made available from a single center. In due course,
the NIH sponsored centralized supply from the US pluripotent
stem cell bank at WiCell in Wisconsin (http://www.wicell.org/).
Although this funding has not been sustained, the operation has
been maintained by WiCell. In the UK, the UKSCB has secured
sustained government support via the MRC and Biotechnology
and Biological Sciences Research Council (BBSRC) and currently
operates a multi-sponsor operation with engagement in research
grants and increasing core support from its host organization the
National Institute for Biological Standards and Control (NIBSC),
now part of the UK regulatory body the Medicines and Healthcare
Products Regulatory Agency (MHRA). In the meantime, other
Government-funded and commercial suppliers of stem cell lines
have been established, and a number of institutions have supported
centers to focus on hiPSC generation and banking (e.g., Rutgers
hiPSC bank, EBiSC, Coriell). Of course, there are many core facili-
ties providing local supplies of stem cells and a few companies who
can provide cells (e.g., Biotime, Cellartis (Takara)) but the number
of public service collections has remained relatively small primarily
due to the reasons outlined above. An additional challenge for
hESC collections has been the ethical debate over the use of human
embryos for research, which requires careful management to assure
the banks operate in an ethically responsible and neutral way.
Since the advent of hiPSC technology in 2009 [6], the ability
to generate stem cell lines using a relatively simple and readily
accessible technique from many kinds of tissue has resulted in a
great increase in the number of iPSC lines. Furthermore, the
capacity to generate lines from individuals with bespoke genotypes
and disease states has led to the development of major programmes
of work to isolate large numbers of lines from patients with inher-
ited disease and other disease states (www.stembancc.org) even to
capture human biological diversity (www.hipsci.org). Details of the
major hiPSC operations are given in Table 1.
These initiatives are currently at an early stage but ultimately,
will deliver a significant resource for research and drug develop-
ment. There are calls for these efforts to be coordinated to make
best use of the resources available and establish common standards,
in part based on the experience and expertise that has been devel-
oped by the stem cell banking field [7].
Table 1
Examples of stem cell banks and core facilities
Distributor Location Website

RCUDR-Infinite Biologics Rutgers University, USA www.rucdr.org
Spanish Stem Cell Bank Grenada, Spain http://www.juntadeandalucia.es/
bancoandaluzdecelulasmadre/
Taiwan Stem Cell Bank Hsinchu, Taiwan http://www.tscb.bcrc.firdi.org.tw//index.do
UK Stem Cell Bank Hertfordshire, UK http://www.ukstemcellbank.org.uk/
WISC Bank WiCell Research Institute/Wisconsin, USA http://www.wicell.org/
Core Facilities (examples only)
Children’s Hospital Boston hESC Core Facility Boston Children’s Hospital/Massachusetts, USA http://stemcell.childrenshospital.org/about-us/
stem-cell-program-labs/the-hesc-core-facility/
Harvard HUES Facility Harvard University/Massachusetts, USA http://www.mcb.harvard.edu/melton/hues/
Harvard Stem Cell Institute iPS Core Facility Harvard University/Massachusetts, USA http://www.hsci.harvard.edu/ipscore/
Stanford Institute for Stem Cell Biology and Stanford University, California, USA http://stemcell.stanford.edu/
Regenerative Medicine
UCONN Stem Cell Core University of Connecticut/Connecticut, USA http://stemcellcore.uchc.edu/
Commercial Banks
Biotime Alameda, USA http://www.biotimeinc.com/
Cellartis (Tokara) Goteburg, Sweden http://www.clontech.com/GB/Products/
Stem_Cell_Research
GlobalStem Maryland, USA https://globalstem.com/
Reproductive Genetics Institute (Stemride International) Illinois, USA http://www.reproductivegenetics.com/
Genea La Jolla, USA http://www.geneastemcells.com.au/
3 Core Requirements for the Establishment of a Pluripotent Stem Cell Bank
There are at least three fundamental issues to address before set-

ting up such a facility:
1. The first step is to clearly identify the primary purpose of the
bank; is it supply of research grade cells, clinical grade cells,
other reagents, training, or a combination of these.
2. Another key decision is whether the bank will operate locally
as a so-called core facility or aim to deliver cells to a much
broader geographical range of clients.
These key decisions will clearly identify the investment required
in staff, facilities, and other resources (for more detailed reviews,
see refs. 7 and 8).
Having set the remit and resources for the Bank, a suitable
infrastructure needs to be established, which should include:
1. An appropriate and robust governance framework. This will
usually comprise the normal institutional management proce-
dures (e.g., health and safety, security, financial accountability)
and the mechanisms for assuring appropriate ethical review
processes (e.g., Institutional Review Board, Local Research
Ethics Committee) are in place to ensure all cell lines are
proven to be isolated from tissues taken with fully informed
and appropriate consent for the distribution of derived cell
lines for any kind of research. In addition, it is important to
have high-quality scientific input by way of an external scien-
tific advisory board or committee to ensure that a bank main-
tains standards and procedures fit for current research need.
2. A system of quality assurance that is suitable for the intended
purpose of the bank and focused on the needs of users groups.
Key to developing this will be the standard of operation set at
the outset (above) as supply for research as opposed to clinical
use will require very different levels of assurance and scrutiny
and compliance with specific quality standards. This will
typically involve the preparation of a high-level operational
manual including key policies and standards and also
documenting all key procedures and protocols and appropriate
record keeping to assure the required level of traceability. This
QA system does not need to be under a formal quality standard
as supply to local researchers could be operated with a minimal
system that suits the clients involved.
The quality assurance system should also include a Cell Line
Master File (employed by the UKSCB) or Cell Line History File as
ref. [3] a key element. This is valuable for both research grade an
clinical grade seed stocks [9, 10], as it can be used to capture all
information from details of informed consent through to quality
8 Glyn Stacey
control data and release of the cell line. Such information may be
difficult to gather retrospectively and will be a critical source of
information to assure appropriate levels of risk assessment, risk
mitigation, and regulatory acceptability of the cell line for clinical
application.
4 Technical Challenges for Stem Cell Banks
In order to deliver the large numbers of existing and future cell

lines, there is a critical need to enhance the efficiency of stem cell
banking and reduce costs. This will require significant develop-
ments in banking procedures and technology. Mechanization and
automation will be the key to this effort and a number of systems
are under development. Improvements to provide stem cell cul-
tures that are consistent and have minimal levels of differentiated
cells will come from improvements in culture media and surface
treatments or 3D culture. In addition, new quality control and
characterization techniques will need to be developed to stream-
line release of cell banks and reduce costs. However, it will be
important to ensure that standards for acceptability of cell lines
are not compromised. Rapid array-based and next-generation
sequencing systems for screening for adventitious agents and
expression of key stem cell markers are already developing that
will need to be qualified for routine use. The use of teratoma
assays is incompatible with such an approach and there are in any
case, serious challenges for the reliability of this type of assay [11,
12] and a comparison of the variation in results fro these assays is
presented in the accompanying tables to ref. [3]. Already array
systems for determining epigenetic status have been proposed for
determining potential pluripotency [13]. However, such assays
will have to be carefully evaluated to ensure that such profiles are
closely correlated with pluripotent capability and will not include
non-pluripotent cell types. It is likely that a rapid cell culture assay
to measure directed lineage commitment will be needed in com-
bination with epigenetic screens. An international collaboration
(the International Stem Cell Initiative; www.stem-cell-forum-net)
is currently comparing such methods with optimized directed dif-
ferentiation protocols and the teratoma assay and is now prepar-
ing its final report (submitted for publication). Hopefully, studies
like this will enable a replacement regime for the teratoma assay to
be established that can enhance the quality and routine use of
pluripotency studies.
4.1 Current Ethical issues remain a challenge in this area. The concerns over
and Developing Issues the use of human embryos to generate hESC lines are often said
for Stem Cell Banks to have been removed by using hiPSC lines. However, it is still
possible that gametes and thus embryos could be generated
in vitro for reproductive cloning using iPSC lines. Public
concern is to be expected wherever work on gametes is proposed

out. The ability to guarantee donor anonymity is also an area for
careful consideration. Large data sets are now available with the
revolution in genomic analytical techniques such as deep sequenc-
ing. Two issues in particular arise from this. One is that research-
ers may be presented with an ethical dilemma should they make an
adverse discovery in a cell line. Appropriate procedures for decid-
ing how to deal with such situations need to be considered care-
fully, with initial regard being given by the Ethics Working Party
of the International Stem Cell Forum [14]. Second, it has been
shown that it is possible to link data in databases with deidentified
genetic data with genealogy websites holding partial Y chromo-
some STR data for known individuals [15]. Many high-level ethi-
cal reviews have concluded that it is appropriate to make scientific
data broadly available to the research community. However, the
mechanisms to assure donors remain deidentified are not secure
against deliberate attempts to reidentify donors and solutions may
have to rely on researchers vigilance and honesty when signing up
to gain access to donor genetic data [16]. In the longer term stem
cell banks and others managing genetic data will need to under-
stand that donor anonymity cannot be guaranteed and consents
must reflect this reality.
A further ethical issues that will challenge stem cell banks, the
whole stem cell field, and society in general are development of
animal-human chimeric tissues in animal models, the capacity of
iPSCs to generate gametes, and potentially embryos and cloned
individuals. More specifically for stem cell banks a further chal-
lenge will be the need to support application of stem cell lines in an
increasing spectrum of research applications.
Automation of stem cell culture processes will be vital to enable
the delivery of the current ambitious large-scale cell banking proj-
ects for iPSC lines. It is currently possible to automate elements of
the cell expansion and characterization tasks but a fully automated
system that does not require manual intervention between recovery
of a vial, its expansion and differentiation ready for final processing
for therapy is not yet qualified for routine clinical use. However,
some groups are moving toward such systems that may be available
for routine use in the future, e.g., Kawasaki, Hamilton, New York
Stem Cell Foundation (NYSCF), Tokyo Electron and Tecan.
Stem Cell research and cell therapy are dynamic and rapidly
progressing areas and it would be easy for stem cell banking centers
to be driven to respond to latest developments that may not be
fruitful in the long run and wasteful of bank resources. It is there-
fore crucial for each banking center to engage high-quality scien-
tific advice that can be used to guide such centers to carry out
appropriate feasibility studies that enable the bank to make secure
decisions that will help to ensure that any major investment in new
technology is effective.
10 Glyn Stacey
References
1. Rottem S, Naot Y (1998) Subversion and setting up and managing a stem cell labora-
exploitation of host cells by mycoplasma. tory. Stem Cell Rev 8(3):830–843
Trends Microbiol 6:436–440 9. Stacey G (2012) Banking stem cells for
2. MacLeod RAF, Dirks WG, Matsuo Y et al (1998) research and clinical applications. Prog Brain
Widespread intraspecies cross-contamination of Res 200:41–58
human tumour cell lines arising at source. Int 10. Stacey G, Masters JR (2008) Cryopreservation
J Cancer 83:555–563 and banking of mammalian cell lines. Nat
3. Andrews PW, Baker D, Benvinisty N et al Protoc 3(12):1981–1989
(2015) Points to consider in the development 11. Buta C, David R, Dressel R et al (2013)
of seed stocks of pluripotent stem cells for clin- Reconsidering pluripotency tests: do we still
ical applications: International Stem Cell need teratoma assays? Stem Cell Res 11:
Banking Initiative (ISCBI). Regen Med 10(2 552–562
Suppl):1–44 12. Müller FJ, Goldmann J, Löser P, Loring JF
4. Andrews PW, Arias-Diaz J, Auerbach J et al (2010) A call to standardize teratoma assays
(2009) Consensus guidance for banking and used to define human pluripotent cell lines.
supply of human embryonic stem cell lines for Cell Stem Cell 6(5):412–414
research purposes. Stem Cell Rev 5(4):301–314 13. Müller FJ, Schuldt BM, Williams R et al
5. Thomson JA, Itskovitz-Eldor J, Shapiro SS (2011) A bioinformatic assay for pluripotency
et al (1998) Embryonic stem cell lines derived in human cells. Nat Methods 8(4):315–317
from human blastocysts. Science 14. Isasi R, Knoppers BM, Andrews PW et al
282(5391):1145–1147 (2012) Disclosure and management of
6. Nakagawa M, Koyanagi M, Tanabe K et al research findings in stem cell research and
(2008) Generation of induced pluripotent banking: policy statement. Regen Med 7(3):
stem cells without Myc from mouse and human 440–448
fibroblasts. Nat Biotechnol 26(1):101–106 15. Gymrek M, McGuire AL, Golan D et al (2013)
7. Stacey G, Crook JM, Hei D, Ludwig T (2013) Identifying personal genomes by surname
Banking human induced pluripotent stem inference. Science 339(6117):321–324
cells: lessons learned from embryonic stem 16. Isasi R (2014) Stem cell research and banking:
cells? Cell Stem Cell 13(4):385–388 towards policy on disclosing research results
8. Inamdar MS, Healy L, Sinha A, Stacey G and incidental findings. In: Dusko I (ed) Stem
(2012) Global solutions to the challenges of cell banking. Springer, New York, pp 29–40
Chapter 2
Quality Assurance in Stem Cell Banking: Emphasis

on Embryonic and Induced Pluripotent Stem Cell Banking
Therése Kallur, Pontus Blomberg, Sonya Stenfelt,
Kristian Tryggvason, and Outi Hovatta
Abstract
For quality assurance (QA) in stem cell banking, a planned system is needed to ensure that the banked
products, stem cells, meet the standards required for research, clinical use, and commercial biotechnologi-
cal applications. QA is process oriented, avoids, or minimizes unacceptable product defects, and particu-
larly encompasses the management and operational systems of the bank, as well as the ethical and legal
frameworks. Quality control (QC) is product oriented and therefore ensures the stem cells of a bank are
what they are expected to be. Testing is for controlling, not assuring, product quality, and is therefore a
part of QC, not QA. Like QA, QC is essential for banking cells for quality research and translational appli-
cation (Schwartz et al., Lancet 379:713–720, 2012). Human embryonic stem cells (hESCs), as cells
derived from donated supernumerary embryos from in vitro fertilization (IVF) therapy, are different from
other stem cell types in resulting from an embryo that has had two donors. This imposes important ethical
and legal constraints on the utility of the cells, which, together with quite specific culture conditions,
require special attention in the QA system. Importantly, although the origin and derivation of induced
pluripotent stem cells (iPSCs) differ from that of hESCs, many of the principles of QA for hESC banking
are applicable to iPSC banking (Stacey et al., Cell Stem Cell 13:385–388, 2013). Furthermore, despite
differences between the legal and regulatory frameworks for hESC and iPSC banking between different
countries, the requirements for QA are being harmonized (Stacey et al., Cell Stem Cell 13:385–388,
2013; International Stem Cell Banking Initiative, Stem Cell Rev 5:301–314, 2009).
Key words Quality assurance, Quality control, Stem cell banks, Human embryonic stem cells,
Induced pluripotent stem cells
1 Introduction
The QA in stem cell banking ensures that the quality of the banked
stem cells is in accordance with national and international guide-
lines and standards for supply of stem cells for research, biotechno-
logical applications, and/or clinical use [1–3]. As such, a bank must
identify the purpose of banked cells to comply with the correct QA
standard to cover the range of potential applications. There must be
a continuous process of managing and evaluating the QA system for
11
12 Therése Kallur et al.
Fig. 1 Schematic of quality management in a stem cell bank
total quality management including risk assessments of all aspects of

stem cell banking (Fig. 1). It includes processes of selection of the
cells donors, the ethical processes around the specific cells banked,
training of the personnel of the bank, the quality and function of
the equipment in the bank, the materials used in culture and freez-
ing of the cells, documentation in the bank, process and material
release, the regulatory standards that are implemented (e.g.,
International Standards Organization; ISO) [4], and the accepted
guidelines and principles in stem cell banking, such as the
International Society for Stem Cell Research (ISSCR) Guidelines
for the conduct of hESC research [5], International Stem Cell
Banking Initiative (ISCBI) Guidance for Banking [1], OECD
Good Laboratory Practice principles [6], as well as appropriate QC.
2 Quality Systems and Standards
Good Laboratory Practice (GLP) regulatory framework is applied

for research grade hESCs, iPSCs, and tissue-derived stem cells
intended for safety studies in animals, while good Manufacturing
Practice (GMP) is required for all clinical grade cells. GLP and
Quality Assurance in Stem Cell Banking… 13
GMP include the quality of management and distribution of the

cell lines as well as the quality of the cells. Of fundamental impor-
tance is that the general ethics principles regarding consent and
donation of human cells and tissue as well as for stem cell banking
have been followed and documented. The cell processing technol-
ogy, management, and running system used in the bank have to be
quality assured. Procedures and protocols for cell culture, freezing,
and thawing must be described and results from cell characteriza-
tion including the ability of the cells to differentiate, their func-
tionality, microbial testing, and tumorigenicity studies must be
sufficiently documented. The bank must have a system for manag-
ing the inventory for assuring credible and accurate storage of
every cell vial, showing the location and number of vials, and who
was responsible for freezing, removing, etc. In addition, specified
testing procedures should be performed before the release of any
cell vials to research and/or clinical recipients.
Examples of QA for clinical grade human embryonic stem cells
have been described previously in the literature [7, 8]. General
standards for QA are applicable to stem cell banking, including
those from the ISO. Such standards are:
1. ISO9001:2000, a general quality management standard for
provision of services and products;
2. ISO17025, laboratory testing and monitoring including the
cell lines for testing of medical products;
3. ISO13485, diagnostic testing procedures including the use of
cells or cell-derived reference materials;
4. ISO34, guide for preparation of reference materials.
The processes and features of stem cell banking requiring QA
are listed in Table 1 and Fig. 2.
Table 1
QA in stem cell banking
Management
Principles of ethical and legal requirements, consents, and agreements
Procedures for receiving, expanding, and storing of stem cells
Operation and maintenance of equipment
Characterization of cells:
(A) Quality and purity of the cell population
(B) Functionality of the cells
• Pluripotency of hESC and hiPSC
• Microbial testing
• Genetic testing
• Tumorigenicity
Release criteria of the cells
Transport and distribution of the cells
Fig. 2 A flow chart of QA in a hESC bank
3 Consents and Legal Framework
Despite several sets of guidelines, the main principles for QA of

stem cell banks are similar in most European countries, North
America, and Australia. The origin of cells is important, together
with the agreements for storage and conditions for distribution.
The stem cell type and the purpose for using the cells varies but the
stem cell bank has to ensure consent by the donating person/per-
sons is voluntary, has been well documented, and guarantees the
privacy of the donor information. Release and distribution of stem
cells should only be possible for projects that are approved by eth-
ics committee and never to any third parties without appropriate
permissions. Commercial and clinical use of the donated stem cells
requires particular ethics and legal agreements that follow the leg-
islation of the country of origin and the country of banking. There
are particular laws for the use of hESCs in almost all countries. The
European laws are presented by European Science Foundation [9,
10]. In addition, the bank should comply with guidelines estab-
lished by the ISSCR [5] which include ethics principles and also
national guidelines such as in the USA [11, 12] (US National
Academy of Science, NAS 2005, NIH Guidelines for Human
Embryonic Stem Cell Research http://stemcells.NIH.gov) and
other relevant regulatory authorities.
Quality Assurance in Stem Cell Banking… 15
4 QA Is Necessary for Stem Cell Banking Activities
In order to ensure the banking and provision of good quality cells,

cell lines must be qualified by mandatory QC procedures for the
banks QA. For example, effective and safe stem cell scale-
up/expansion methods must be employed to avoid culture adapta-
tion or tumorigenic mutations. Avoiding animal-derived components
in the cultures or feeder cells is a preferred strategy to humanize and
simplify processing. If feeder cells are used, they should be of human
origin and ideally GMP or GLP grade depending on the sown-
stream application [13, 14]. It is advisable to keep the passage level
of the banked stem cells as low as possible in the master cell bank
(MCB), which maximizes up-scaling and supply of more cells of a
particular passage. Furthermore, it is advisable that the stem cell
bank use cell cultivation methods that are considered current best
practice. According to our experience, the use of human recombi-
nant laminin-521 as culture substrates and chemically defined cul-
ture media allow the effective expansion of hESCs [15].
Importantly, optimized phenotype characterization methods
for each stem cell type are essential, including testing for pluripo-
tency in vivo by teratoma test in immune-compromised mice, and
in vitro by differentiating them into mesoderm, endoderm, and
ectoderm derivatives. In order to confirm pluripotency, cells should
exhibit typical cell surface marker profile by the expression of rel-
evant proteins suing, for example, flow cytometry and
immunocytochemistry.
Exclusion of tumorigenic changes and abnormal karyotype is
an essential part of safety testing. This can be done using standard
G-band analysis, comparative genomic hybridization (CGH), and
single nucleotide polymorphism (SNP) testing [16–19] but also by
high-throughput sequencing.
Microbial testing is also an important part of QC and the
release criteria encompassed by the bank QA. Microbial contami-
nation not only affects the quality of the research data obtained
from a particular cell line but also poses a risk for staff at the bank.
The QA system must include test criteria for cell donors, cell lines,
and reagents and biological material used for cell culture (see
Chapter 6).
An important part of running a cell bank is ensuring that the
quality and identity of the cells are maintained during the transport
and distribution of the cells to and from the bank. This should
include having standard operating procedures (SOPs) in place for
temperature control, as wells as for record keeping including deposit
and withdrawal or requisition of cells. For cells intended for clinical
use, a guidance for how such a system may be established can be
found in the EU guidelines on Good Distribution Practice (GDP)
of medicinal products for human use [20]. Stem cells can be used
in research or in clinical treatment. Given the many different stem
cell types and grades, it is important for the end users to know
exactly which stem cells are being provided for their particular pur-
pose. There are specific requirements and standards for cells for
both research and clinical use, including sterility, functionality,
genetic stability, lack of immunogenic substances, traceability of the
culture constituents, in addition to traceability of the cells and cell
donors. Ensuring the requirements are met will ensure safety, qual-
ity, efficacy, and reproducibility.
References
1. International Stem Cell Banking Initiative 12. National Institutes of Health (NIH) Guidelines
(2009) Consensus guidance for banking and for Human Embryonic Stem Cell Research.
supply of human embryonic stem cell lines for http://stemcells.NIH.gov
research purposes. Stem Cell Rev 5:301–314 13. Unger C, Skottman H, Blomberg P et al
2. Crook JM, Hei D, Stacey G (2010) The (2008) Good manufacturing practice and clin-
International Stem Cell Banking Initiative ical grade human embryonic stem cell lines.
(ISCBI): raising standards to bank on. In Vitro Hum Mol Genet 17(R1):R48–R53
Cell Dev Biol Anim 46:169–172 14. Prathalingam N, Ferguson L, Young L (2012)
3. Healy L, Young L, Stacey GN (2011) Stem Production and validation of a good manufac-
cell banks: preserving cell lines, maintaining turing practice grade human fibroblast line for
genetic integrity, and advancing research. supporting human embryonic stem cell deri-
Methods Mol Biol 767:15–27 vation and culture. Stem Cell Res Ther
4. The International Organization for 3(2):12
Standardization. www.iso.org 15. Rodin S, Antonsson L, Niaudet C et al (2014)
5. Guidelines for the Conduct of Human Clonal culturing of human embryonic stem
Embryonic Stem Cell Research (2005) www. cells on laminin-521/E-cadherin matrix in
ISSCR.org defined and xeno-free environment. Nat
6. OECD (2013) Principles of good laboratory Commun 5:3195
practice and compliance monitoring. www. 16. Hovatta O, Jaconi M, Töhönen V et al (2010)
OECD.org A teratocarcinoma-like human embryonic
7. Crook JM, Peura TT, Kravets L et al (2007) stem cell (hESC) line and four karyotypically
The generation of six clinical grade human normal hESC lines reveal high oncogenic
embryonic stem cell lines. Cell Stem Cell potential. PLoS One 23(5):e10263
1:490–494 17. Närvä E, Autio R, Rahkonen N et al (2010)
8. Ilic D, Stephenson E, Wood V (2012) High resolution genome wide DNA analysis
Derivation and feeder-free propagation of on a large panel of human embryonic stem
human embryonic stem cells under xeno-free cells reveals novel genomic changes associated
conditions. Cytotherapy 14:122–128 with alterations in gene expression. Nat
Biotechnol 28(4):371–377
9. Hovatta O., Walles H., Agovic A. et al.
(2010) Human stem cell research and 18. International Stem Cell Initiative (2011)
regenerative medicine: European perspec- Screening ethnically diverse human embryonic
tive on scientific, ethical and legal issues. stem cells identifies a chromosome 20 minimal
ESF Science Policy Briefing 38. http:// amplicon conferring growth advantage. Nat
www.esf.org/publications/science-policy- Biotechnol 29:1132–1144
briefings.html 19. Stephenson E, Ogilvie CM, Patel H et al
10. Hovatta O, Stojkovic M, Nogueir M, Varela- (2010) Safety paradigm: genetic evaluation of
Nieto I (2010) European scientific, ethical and therapeutic grade human embryonic stem
legal issues on human stem cell research and cells. J R Soc Interface 7(Suppl 6):S677–S688
regenerative medicine. Stem Cells 28:1005–1007 20. Information from European Union institu-
11. US National Academy of Science (2005) tions, bodies, offices and agencies. Other Acts.
Guidelines for Human Embryonic Stem Cell Guidelines of 7 March 2013 on good distribu-
Research. www.nap.edu tion practice of medicinal products for human
use (2013/C 68/01)
Chapter 3
Acquisition and Reception of Primary Tissues, Cells,

or Other Biological Specimens
Lyn E. Healy
Abstract
The use and banking of biological material for research or clinical application is a well-established practice.
The material can be of human or non-human origin. The processes involved in this type of activity, from
the sourcing to receipt of materials, require adherence to a set of best practice principles that assure the
ethical and legal procurement, traceability, and quality of materials.
Key words Tissue, Cells, Biospecimens, Procurement, Consent, Quality, Best practice
1 Introduction
There is a wide variety of biological material used in the processing

and banking of stem cells. However, not all material is of human
origin and not all contains viable material. This chapter primarily
focuses on human material but also considers other sources of
material frequently used both in and ancillary to the banking pro-
cess. The sourcing, procurement, acquisition, and receipt of human
biological material for research or clinical application form the ini-
tial key stages in the activities of stem cell banking and bioprocess-
ing [1–3]. The material may be used for a number of activities:
hemopoietic stem cells (HSC) for clinical application [4], various
sources of tissue as starting material for the generation of cell lines,
including induced pluripotent stem cells (iPSCs) [5, 6], other bio-
logical material for example extracted nucleic acids used for controls
in routine molecular biology. Ensuring that cells, tissues, cell lines,
and other biological material are sourced and procured ethically and
in accordance with the local legal framework underpins the operat-
ing principles of any reputable biorepository and the activity of stem
cell banks falls within this category of repository. Human stem cell
banks are repositories that either specialize in the banking of a par-
ticular cell type or bank a number of cell types including somatic
primary cells (e.g., cord blood, mesenchymal, embryonic stem cells,
17
18 Lyn E. Healy
and iPSCs [7–17]). Such repositories operate within a governance

framework to provide ethically procured, quality controlled cells
for either research or clinical application or both [18–25]. The
impetus behind the establishment of these resources is to provide a
central source of good quality material. The banks can range in size
from small banks with a limited range of biological material to
large commercial banks with a wide range of material available in
quantity.
Once a source of human biological material has been identi-
fied, the issue of appropriate consent needs to be addressed, to
ensure that the tissue or cells identified can be used for their
intended application. A chain of custody needs to be established to
enable traceability from procurement to receipt into the bioreposi-
tory or cell processing laboratory. This traceability is required for
Good Manufacturing Practice (GMP) and is a principle of both
good laboratory practice (GLP) and good cell culture practice
(GCCP) [26, 27]. The biosafety of the biological specimens is also
a prime consideration as it is important to know that the materials
can be handled safely in a laboratory setting and if applicable, can
be utilized in the clinic.
The basic operational principles for biorepositories are derived
from Best Practice Guidelines and consensus documents [28–34]
that are periodically updated to incorporate changes in policy,
legislation, improved application, and the evolving areas of
research, all of which have an impact on the work of the reposi-
tory. Although these documents focus primarily on research
repositories, the same principles are applied to clinical reposito-
ries, since the operating principles are in alignment. However, in
the clinical setting, additional requirements may need to be incor-
porated into the operation of the repository to include local,
national, and international standards for clinical materials [4, 35].
The guidelines cover repository planning, facilities, document
control, storage, equipment, safety, training, working environ-
ment, record keeping, quality control, quality management, and
termination of repositories.
This chapter outlines the high-level principles of best practice
for the sourcing, procurement, acquisition, and reception of bio-
logical materials into a laboratory for cell processing and stem cell
banking purposes.
2 Sourching Material for Procurement and Acquisition
2.1 Sourcing Human When sourcing biological material, the first question that needs to
Primary Tissue, Cells, be asked is what is the intended purpose of the tissues, cells, or
and Biological other biological material? Is the material for clinical application or
Specimens for research? The latter question determines how the procurement
process will be undertaken. For both clinical and research applica-
tions, it is essential that the primary biological material has been
Acquisition and Reception of Primary Tissues, Cells, or Other Biological Specimens 19
consented for the intended purpose [36–38]. The consent needs

to have been informed and when initiating procurement this
should always be a fundamental part of the due diligence process.
Always ensure that full disclosure of the actual and potential use of
materials has been addressed, for example, donation of material for
clinical application, export of material, potential commercial use of
material, or publication of genetic data from the material. In addi-
tion, any limitations on the use of the material should also be
noted.
Suppliers of primary human material are often tissue banks
[39], located within a hospital or other medical setting, thereby
facilitating both access to material and an ethical review mecha-
nism and ensuring appropriate governance of local practices. On
the other hand, there are suppliers independent of the medical set-
ting that procure ad hoc for bespoke requirements. However, all
suppliers are regulated by national and international guidelines and
legislation and this regulation will depend on the type of procure-
ment being carried out. In the case of clinical application [40–42]
in the United Kingdom and the United States [35], it is suggested
that for donation and procurement, a number of points should be
considered and procedures put in place. For donation, good clini-
cal practice (GCP) should be followed with respect to consent,
ethics, donor screening and history, and payment for donation.
With procurement, post-donation, there should be procedures set
up for the handling of material, suitable storage facilities, appropri-
ate labeling (an anonymized coding system), and tracking to ensure
traceability and a system to deal with discarded tissue.
When primary human material is used for research, this activity
is again controlled by local, national and international legislation,
regulations, and guidelines [43]. In the research setting, in gen-
eral, any project involving the use of primary tissues or cells will be
subject to an ethical review process prior to the initiation of that
project, to ensure that the rights of the donors are protected and
that the work for which the tissues or cells are used is of benefit to
the donor, to society, and to science.
The procurement of cells for the derivation of human embry-
onic stem cells (hESCs) for research or clinical application is sub-
ject to local legislation that can be both complex and involve a
highly regulated infrastructure to ensure the ethical provenance
and use of this ethically sensitive material [43–45].
Specimens of primary human tissue are used in stem cell bank-
ing often as controls in quality control (QC) and other specific
in-process assays. These materials are also subject to local and
national guidance and legislation. Again, consent is required and
this should be informed and the material should only be used for
the purpose for which it was consented. Procedures for procure-
ment, receiving, handling utilizing, and disposing of the tissue
should be established enabling traceability, although the samples
should be anonymized.
20 Lyn E. Healy
2.2 Sourcing hESC It is fundamental best practice that cell lines should always be
and iPSC Lines sourced from a reputable biorepository [46, 47]. This ensures a
consistent, reliable, and replenishable supply of cells, adding robust-
ness and reproducibility to any system and process that utilize the
cells [26, 48, 49]. In general, human cell lines obtained from rec-
ognized cell banks tend to be tested for blood-borne viruses and
are free from bacteria fungi and mycoplasma. It is also common for
the lines to be subjected to DNA profiling via Short Tandem
Repeat (STR) [50–53] analysis to enable authentication and facili-
tate cell line identity in the event of cross contamination [54, 55].
The production of the first hESC line in 1998 heralded an era
of promise for the generation of a plethora of cellular therapies.
There are in excess of 1000 hESC lines available [56–58]. Much
research has been generated from these lines. However, the lines are
ethically contentious [59] and may be subject to stringent domestic
laws. In order to minimize the number of embryos used to generate
lines, a number of countries have set up banks to store and distrib-
ute hESC lines for research purposes [60]. In order to provide a
coordinated approach to the distribution of hESC lines internation-
ally, the International Stem cell Banking Initiative (ISCBI) under
the auspices of the International Stem cell forum (ISCF) [61] pro-
duced a consensus guidance for the banking and supply of human
stem cell lines for research purposes [62]. This covers all aspects of
stem cell banking including consent, governance, characterization,
and QC. With respect to consent, in the UK a network of hESC
coordinators was set up to develop best practice models for hESC
derivation and have produced a national consent form that has been
used extensively in the research setting [63].
As the field of hESCs moves toward therapeutic use, the tech-
nology and regulatory framework is in place to facilitate the
generation of hESCs for clinical application [45, 64] and a number
of stem cell lines have been generated for this purpose [65–68]. In
addition, a number of banks have been accredited nationally for
the banking and storage of hESCs for clinical application. The
ISCBI has formulated a “points to consider” document for the
banking of human stem cell lines for clinical application [69].
In 2007, the production of human iPSC from fibroblasts was
described [70]. The iPSCs were generated by reprogramming
somatic cells to ES-like cells using four transcription factors [71–
74]. Since this type of cell is derived from somatic cells it is not
ethically contentious; however, appropriate consent for intended
use is still a prerequisite for the production of a cell line from the
primary tissue [75–81]. The cells can be generated for use in the
clinical setting and are potentially useful for autologous transplan-
tation (personalized medicine) to treat the donors from which the
primary tissue was isolated, thereby alleviating potential immuno-
genic reactions [82–84]. There are now many iPSC lines [85, 86]
being generated using a variety of methods for reprogramming
[71–74] and as with other cell lines these lines should be obtained
from a recognized supplier. The principles outlined in the consen-

sus document for hESCs [62] are applicable to iPSCs.
2.3 Other Primary The coculture of cells during the process of stem cell banking is a
Tissues and Cell Lines common practice, especially in the case of pluripotent stem cells
(PSCs). Fibroblasts derived from human or murine sources are
inactivated and used as feeder cells to support the growth and
expansion of PSCs. The procurement of the human tissue from
which these cells are derived is covered in Subheading 2.1. In the
case of animals the material should be traceable to an animal col-
ony from an ethical animal resource where best practice in animal
husbandry is executed. The use of animals in research is regulated
in many countries [87] and local legislation could apply to the
procurement of these tissues.
As stated in Subheading 2.2 cell lines should always be sourced
from reputable cell providers [46, 47] to assure attributes such as
supply, provenance, and quality control. During the banking pro-
cess, established animal cell lines and genetically modified cell lines
might be used for QC applications such as positive or negative
phenotyping/genotyping controls, biosafety assays (which could
include infectious material for controls), surrogate assays, etc.
2.4 Other Biological Apart from tissues and cells, the stem cell bank might procure
Specimens Used other biological specimens for use in activities such as process qual-
in the Stem Cell ification/validation, quality control, assay development, service
Banking Process provision, and bespoke testing and banking. These may take the
and Related Activities form of purified or partially processed products such as isolated
nucleic acids, proteins, cell lysates, or raw products such as blood
or other body fluids from which specific biological products could
be isolated. Again, depending on the source and the level of manip-
ulation these materials may fall under a regulatory framework.
3 Points to Consider with Respect to Procurement from Suppliers of Primary

Tissues, Cells, Cell Lines, and Other Biological Specimens
When sourcing a supplier of biological material it is good practice

to undertake a due-diligence exercise, to ascertain whether the
supplier is fit for purpose. The exercise should address but not be
limited to the following: (a) Is the supplier licensed for regulated
activities? (b) Does the supplier have a quality management system
in place? (c) Does the supplier subcontract? (d) How does the sup-
plier control any subcontracting, (e) Does the supplier have an
internal audit system? (f) Does the supplier perform QC testing?
(g) Does the supplier perform safety testing? The application of
this type of process will enable a customer to select a supplier suited
to the specific requirements of the banking activities.
22 Lyn E. Healy
A material transfer agreement (MTA) might be required to be

put in place between the supplier and the biorepository. This will
set out the terms and conditions for the deposition of the biologi-
cal material in the bank and might include such elements as owner-
ship, intellectual property rights, restrictions on the distribution of
material, and any licencing terms for the use of the material.
4 Accessioning and Receipt of Biological Materials

into the Stem Cell Banking Facility
As biorepositories, stem cell banks are dedicated facilities, often

licenced for specific activities. The repository should adhere to
ethical principles recognized internationally since they deal with
sensitive material and data. The bank needs to assure the traceabil-
ity of the material and ensure the quality of the material. The labo-
ratories located within the repository should be designed to be fit
for purpose [1, 28, 30, 88, 89] and the staff formally trained to
process and document the biomaterials handled in the facility.
Accessioning biological materials into a bank comprises several
processes including: (a) transportation between supplier and recip-
ient, registering receipt of the material, (b) checking and recording
all documentation related to the material, and (c) storing the mate-
rial appropriately upon receipt. However, prior to accessioning and
the initiation of the procurement process a risk analysis of the activ-
ity of the bank should be undertaken to enable suitable docu-
mented risk assessments to be prepared for the handling and
processing of the biological material [28, 30]. Once a supplier of
biological material has been selected and the procurement process
has been initiated, then the risk assessment can be completed prior
to the receipt of the material. Human material could potentially be
infected with micro-organisms or viruses, which may be patho-
genic in nature. Banks should ensure that they are capable of han-
dling and disposing of biological material at the appropriate level
of containment to ensure that the safety of the staff is not compro-
mised in the workplace. Following a successful procurement, the
biological material needs to be transported to the bank.
The bank will have a Standard Operating Procedure (SOP)
for transportation of materials for receipt into the bank. The sup-
plier, as the shipper, will have determined how the biological
materials should be transported so that the packaging and ship-
ping complies with national and international standards, guide-
lines, and regulations. Biological material is categorized, in
general, as dangerous goods and therefore requires all staff
involved in the shipping of these materials to be suitably trained.
Local, national, and international regulations relating to the mode
of shipping these materials should be adhered to [90, 91].
On receipt of material into the repository, the material should

be handled and stored in a safe and appropriate manner, as dictated
by the documented risk assessment. The material should be given a
unique accession number to facilitate the tracking of the material
through the processes of storage, utilization, and/or distribution
and disposal. It is best practice not to reassign the accession number
to any other biological material even when the original material has
been removed from the bank or disposed of. This will avoid any
confusion between the biospecimens in tracking systems. All docu-
mentation from the supplier of materials should be checked. Any
information that could lead to the identification of the donor should
be removed to prevent association between the donor and the
material supplied. This documentation may contain such informa-
tion as MTAs, consent, cell line information, testing for pathogens,
and karyotyping data. The accompanying paperwork should be
linked to the accession number and this number should be present
on all subsequent documentation related to the material, thereby
facilitating traceability throughout the life cycle of the material.
The material will then enter the management system of the
biorepository and will be dealt with as per local SOPs or in accor-
dance with specific regulatory procedures. These procedures may
include, if appropriate, the quarantine of new biological material
and should include quality assessments, such as the authentication
and characterization of materials [28, 30, 31, 52, 55, 62]. Best
practice encourages the provision of a series of process manage-
ment documents covering all aspects of the life cycle of the material
including legacy planning [92].
In conclusion, as the demand for biological materials increases
[93, 94], the role of biobanks becomes more important as reliable,
ethical resources for well-characterized biospecimens. International
harmonization and standardization of best practice in this area will
facilitate and normalize the sourcing, acquisition, and reception of
these biomaterials into the biorepositories. This in turn will enable
the development of a global resource of consistent biological mate-
rials for research and clinical application.
Acknowledgments
The author would like to thank the Medical Research Council

(MRC), the Biotechnology and Biological Research Council
(BBSRC), the Technology Strategy Board (TSB) the EU
Framework 7 programme projects; Embryonic Stem cell-based
Novel Alternative Testing Strategies (ESNATS), Stem cells for rel-
evant efficient extended and normalized toxicology (SCR&TOX),
ToxBank and the International Stem Cell Forum for supporting
the activities of the UK Stem Cell Bank.
24 Lyn E. Healy
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Chapter 4
Information Management
Alberto Labarga, Izaskun Beloqui, and Angel G. Martin
Abstract
The collection and storage of human tissue samples has been undertaken in medicine for centuries; how-
ever, biobanking has only recently become a dedicated activity. The technological developments that have
allowed the procurement and long-term storage of viable human cells ex vivo, and to obtain relevant sci-
entific information, including genetic information, provide tremendous possibilities for advancing bio-
medical research. At the same time, these possibilities have raised complex information management issues
regarding samples, processing, donor information, traceability, and use of the sample. This chapter consid-
ers the requirements for managing information within biobanks, critical to their operation. Special consid-
eration is given to Laboratory Information Managing Systems (LIMS) as a tool for comprehensive access
and storage of information.
Key words Laboratory Information Managing Systems, LIMS, Biobank, International Standards
Organization, ISO
1 Introduction
Population-based biobanks aim to recruit high-quality samples from

a large number of individuals, associated with detailed epidemiologi-
cal, genealogical, lifestyle, and clinical data. A common goal is to
support research projects relating genetic/genomic factors underly-
ing complex, multi-factorial human diseases and to investigate how
interaction between genes and environment affects our health.
Harmonization of sample collection procedures, technical process-
ing, quality standards, and the type of information recorded is
important, providing for international collaborative research involv-
ing large numbers of subjects, where generalizability of findings
across populations can be made. For such studies, it is of vital impor-
tance to establish quality criteria concerning the nature of the sam-
ple, conditions of storage, and adequacy of available information.
The primary objective of biobanking is to provide a large, well-
annotated repository of biological samples for scientific research.
Although the storage of human biological material such as tissue
29
30 Alberto Labarga et al.
and/or cells is a prerequisite, the information generated from han-

dling and analyzing the tissue (genotype) and about the tissue
donors is vital. So the collation and provision of quality informa-
tion is a principal and significant undertaking for biobanks.
Information management is often specific for a particular insti-
tution. It is therefore desirable to enable the exchange of informa-
tion between networked biobanks without compromising core
services and security through, for example, web integration with
web service standards being incorporated in the system applied.
A biobanking information management system should consist of at
least two tools: (a) a complete database containing all information
associated with a biological sample and (b) an extraction tool (pref-
erably web based) [1].
Any informatics solution must be designed to facilitate proper
biospecimen storage and handling and regulatory compliance
(including certification, e.g., ISO, good laboratory practice [GLP],
good manufacturing practice [GMP]), and to increase the value of
the biorepository by providing biobank personnel and researchers
with clinical, genomic, proteomic, sample procurement, and other
information.
2 Information Flow in a Biobank
In keeping with the different types of biological samples used in

research and therapeutics, there are several types of biobanks.
However, they all face similar challenges related to information
management. Any information system designed for a biobank
should therefore address the following [2]:
2.1 Sample The details of how the sample was obtained and from whom,
Procurement including personal and/or clinical data of the donor, must be cata-
logued. Sample information is usually presented in the form of
electronic documents that are categorized and have appropriate
access control. Information regarding patient consent can be used
to demonstrate that appropriate approval was obtained prior to use
of a particular sample. Sample deidentification or coding should be
performed.
User accounts and roles define security access rights, ensuring
that only the users with sufficient access privileges may access sensi-
tive information.
2.2 Sample Depending on the type of biological material to be banked, the sam-
Processing ple will require simple unprocessed storage to complex multi-step
precessing. The latter may involve extraction of DNA, RNA, pro-
tein, particular cell types, and/or cell culture using SOPs with record
keeping. Informatics systems may need to be designed ad hoc, so
the information can be readily recorded for any particular situation.
Information Management 31
2.3 Sample Storage Storage locations need to be predefined, with samples appropri-
ately contained in a box, bag, multi-well plate, or cryovial. Bar-
coding of containers may be applied. Importantly, sample storage
entails:
1. Finding a suitable storage location with the information man-
agement system providing the best sample location based on
criteria such as availability of space, sample pooling, accessibil-
ity, and predictive time of storage.
2. Identifying a most suitable sample (with pull lists) requested.
3. Retrieving a particular stored sample.
2.4 Sample A sample may need to be relocated from its original location to a
Movement new one as the biobank grows, in case of equipment failure, or to
rearrange samples for more efficient storage. Therefore, the infor-
matics system must track the movement of samples within the bank.
Storage systems can vary, from simple shelving to complicated bar
code assisted plate storage in climate-controlled facilities.
2.5 Sample Shipping Information such as transport conditions, specimen expiry date,
shipping dates and instructions, sample type, research project, and
billing requirements should be collated.
2.6 Chain of Custody Most biobanks incorporate the role of Data Manager who is
responsible for the custody of the biobank data files, including
donor information. The position is responsible for granting bio-
bank data accession (e.g., to regulatory authorities, health authori-
ties, researchers, donors, etc.). The position may utilize an LIMS
to facilitate data management and ensure as a legal requirement
that the “biobank file” be registered with a corresponding agency
for protection (AEPD in Spain), therefore ensuring proper treat-
ment of information.
3 Laboratory Information Management System (LIMS)
LIMS is a laboratory and information management system with

features that support tracking and organization of biological sam-
ples, tests results, methods, specifications, access control, etc. Many
organizations consider their LIMS to be a mission-critical compo-
nent of their overall corporate information system, whether the
company is in a regulated industry, such as drug development or
pharmaceutical manufacturing, or in a nonregulated industry, such
as petrochemicals. LIMS provides the integral connections to these
companies’ laboratory data, instruments, analysis, and reports.
A major advantage of LIMS is improved time management
(e.g., by more efficient sample login), reporting, and data quality
and security. An LIMS analyst can pull names from a list with cell
line specific information and QC already set up; thus, the informa-
tion does not have to be reentered each time. In addition, there is
complete chain of custody and a full audit trail, work lists can be
generated as well as backlog and production reports, and a sam-
ple’s status can quickly be determined at any time.
After sample login, the system can automatically generate a
report that can be printed, emailed, or displayed on a web site in a
read-only format. Furthermore, data quality can be enhanced by
verifying the data format, providing an audit trail, and reducing
data entry errors. Data search time is decreased by including simple
pull-down lists, and QC is supported by automated data entry and
validation, with instrument integration (i.e., analysts do not have
to manually enter results from instruments).
Selecting an LIMS requires that the system matches the labo-
ratories sample flow, while being flexible enough to accommodate
change, with an upgrade path being available as the laboratory
grows. Therefore, as a laboratory’s data management and report-
ing needs expand, a clear migration path so that none of the cus-
tomizations previously made are lost.
The LIMS may also need to interface with other databases,
such as accounting, inventory, or material safety, which increases
the importance of selecting a system that is Open Database
Connectivity (ODBC) compliant. ODBC is a database standard
that provides the ability to link the LIMS with different databases.
LIMS may also connect the analytical instruments in the labora-
tory, so it must support RS-232 connections, automatic polling for
files, and a flexible parsing interface that can be easily configured to
support the different data formats generated by the instruments.
Plate management capabilities and interfacing with liquid handling
robots are other features commonly found in modern LIMS that
may be useful for biobanking.
The first commercial LIMS was introduced and developed in
the 1980s by analytical instrument manufacturers. The system was
quickly followed by second generation LIMS, which used relational
databases (RDB) to provide application-specific solutions. The
increase in computer-processing speed, the enhancements in third-
party software capabilities, and the reduction in PC, workstation,
and server costs paralleled the introduction of the commercial LIMS.
The advantages resulted in a shift away from proprietary commercial
systems toward open systems that emphasized user-configurability,
rather than customization by the vendor. By the time the third-gen-
eration technology was introduced in the 1990s, LIMS combined
the PC’s easy-to-use interface and standardized desktop tools with
the power and security of minicomputer servers in a client–server
configuration. That is, its architecture splits the data processing
between a series of clients and a database server that runs all, or part
of, the relational database management system. When the Internet
took off in 1996, the first web-enabled LIMS was soon introduced,
followed by web-based and thin-client solutions. Understanding the

differences between these architectures is challenging because the
terms are often used interchangeably, making it difficult to choose
the “right” LIMS. This choice, in turn, affects the cost of clients and
servers, third-party software needed to connect these LIMS clients,
and the resources needed to deploy and upgrade the LIMS.
Furthermore, it affects the robustness and security of the application
as a whole, as well as the flexibility of the system for later modifica-
tion. Depending on the answer and the tradeoffs to be made, the
organization may decide to use either a thin-client, web-based, web-
enabled, or a thick-client solution (Fig. 1).
Thick-client is used to describe an application designed to run
in a client–server environment, meaning that a portion of the soft-
ware resides on the server, and the other portion resides on the
user’s workstation. Clients are essentially the PCs or workstations.
It is the client that performs the bulk of any data-processing opera-
tion and relies on its associated server primarily for data storage.
A thick-client has the LIMS installed on the PC’s hard drive. The
thick-client connects to the database server, but the processing is
done on the client side (Fig. 1a). Each change to the configuration
or the application must take place at the client level; that is, the
modifications must be propagated to each individual workstation.
Web-enabled is used to describe the add-on web browser com-
ponent of an application designed to run in a client/server envi-
ronment. The web-enabled portion of the application may allow
access to data from a web browser, but the user is limited to the
product functionality that is available on the web portion of the
system (Fig. 1b). To access and exploit the full functionality of the
application, users must have the local client installed on their
desktops.
Thin-client or web-based applications offer end-users full
application functionality from a browser. A thin-client does not
have significant hard drive or memory requirements, as it is usually
a simple web browser that does not store or process data. The
LIMS resides on the application server(s) while the thin-client sim-
ply presents the screen display and allows users to interact with the
application server via a keyboard and a mouse (Fig. 1c).
Configuration changes or customizations made on the server to
the software are immediately universally available to all end-users.
Thick clients and web-enabled solutions present a series of
advantages. Typically, they require less network bandwidth.
Because thick-clients themselves do much of the application pro-
cessing, they do not require an application server for processing.
However, it is important to remember that the database will likely
reside at the server level, so communication to and from each
thick-client is still required. They can also cope with applications,
which require a significant amount of computing power, such as
chemical drawing, image processing, or molecular modeling.
Fig. 1 Possible LIMS configurations. (a) A thick-client connects to the database server, but the processing is
done on the client side. (b) For a web-enabled architecture, users access the LIMS from their browsers, but the
system has to be installed on the server. (c) Web-based architectures move the logic of the system to a web
application that can be accessed from a browser
Besides, thick-clients provide some level of redundancy. In a

simple LIMS solution that uses a single thick-client, if the server–
network goes down, the client can still collect sample data and
hold it on the PC’s hard drive until recovered data can be for-
warded to the appropriate server. In a thin-client application,
redundancy is achieved through clustered application servers that
provide load balancing and fail-over, a process that routes client
requests to servers within the cluster. If one or more servers fail,
client requests are automatically routed to other servers within the
cluster so there is no break in service. But this increases the cost of
the overall systems in terms of hardware, software licenses, and
maintenance.
The main advantage of thin-client LIMS solutions is that users
can access the LIMS from virtually any Internet-ready device. All
of the laboratory’s applications and data are maintained centrally,
thus allowing any number of people to share them in a secure way
by simply plugging in a thin-client browser. Thin-clients also
enable connectivity by critical users external to the laboratory such
as executives, customers, and partners. With thin-clients, all the
applications and data are maintained on a central server. For lead-
ing thin-client LIMS, access is role-based and password-protected
for security, complying with 21 CFR Part 11. Disabling a login
account disables access to all company information. No application
data ever resides on the client; hence files cannot be transferred to
local hard drives and memory sticks.
To ensure successful management of the LIMS implementa-
tion and maintenance, selecting the best LIMS architecture for an
organization is more critical than ever. But with the decreasing
hardware and software costs and growing acceptance of the
Internet, the time for laboratories to move from paper or spread-
sheet tracking systems to an LIMS has never been better [3].
4 Key Points on Implementing a LIMS in a Stem Cell Biobank
Based on our own experience with setting up a LIMS in a stem cell

biobank, we have identified a number of issues that may need to
be defined up front when implementing a LIMS solution. It is key
to having a clear idea of the critical points that the LIMS will
address, as the more defined the processes to be controlled are
from start, the more affective the design of the system will be.
Otherwise, changes in the workflows down the road may become
necessary, which may be costly and time consuming, especially if a
custom solution is selected. Nevertheless, the LIMS must allow
enough flexibility to accommodate future changes in the scope
and operation of the biobank. For a list of available LIMS manu-
facturers, see Table 1.
36
Table 1
Main LIMS vendors for data management in biobanks
Vendor Main features Architecture References in biobanking

Alberto Labarga et al.
Labware LIMS More than 500 modules available to licensed Thick-client and web Karolinska Institute (Sweden)
http://www.labware.com/ users offers flexibility that covers everything enabled University of Connecticut
the user may need, including basic scientific Inbomed (Spain)
document management. Instrument ONT (Spain)
integration using Labstation. Labware
offers a separate ELN product that
seamlessly integrates with the main LIMS.
StarLIMS Three separate components (LIMS, SDMS, A special version of a VA Biobank (USA)
http://www.starlims.com/ and ELN) that can be fully integrated and thick-client that runs on a UK Biobank
accessed from a browser. browser using Microsoft.
Net framework
Labvantage Saphire LIMS Separate offer for LIMS and an ELN Web based Copenhagen University BioBank for
http://www.labvantage.com/ (eNotebook). First vendor to offer a Experimental Research
web-based solution. Easy to configure and International Genomics Consortium
maintain. Human Cancer Biospecimen Core
Resource
Nautilus LIMS Most diverse offer, with five different LIMS Thick client Hunt Biobank (Norway)
http://www.thermoscientific.com and many other products licensed Singapore Tissue Network
separately.
1. Scope Definition
First, it is important to clearly state all activities required
to be covered. Such activities may include:
● Wet laboratory workflows.
● Sample cession.
● Performance metrics.
● Reports.
2. Sample workflow management.
The information that may need to be recorded upon sam-
ple reception is:
● Login details:
– Login date
– Internal ID
– Receipt by
– Sample type
– Sample handling considerations
● Donor information (minimal information about the
patient)
– Donor type
– Age
– Sex
● Sample collection
– Date and time
– Medical center
– Preservation solution
● Incidents
3. Cell collection and isolation details
● Technician
● Date
● Number of cells
● Cell viability
● Cell seeding density
● Quality control information
4. Primary cell culture
● Technician
● Date
● Number of cells
● Cell viability

● Number of frozen vials at stage 1 (BCM)
5. Secondary cell culture
● Technician
● Date
● Number of cells
● Cell viability
● Number of frozen vials at stage 3 (BCT)
6. Cryopreservation:
● Input date
● Freezing process data (if a controlled-rate freezer is used)
● Output date
● Technician
7. Liquid nitrogen (LN2) storage
8. Sample provision
● Information about the petitioner organizations
● Vials to be provided, with exact localization and unit price
● Authorization by quality manager
● Authorization by biobank supervisor
● Report generation (delivery note and invoice)
9. Performance metrics
● Annual production and historic production data
10. System architecture
A modular architecture is desired to introduce the flexibil-
ity required to manage the constant evolution of the biobank
needs. Whenever the biobank has to adapt its processes, the
modules, fields, and items need to be modifiable, as well as
rules and work cycles of the LIMS.
11. Dataflow Key Points
The large amount of data that the user has to input can be
overwhelming, so it is important that the system clearly identi-
fies which are the mandatory pieces of data, not allowing the
user to continue until they are fully fulfilled. This ensures the
overall quality of the biobank and that all samples comply with
given specifications.
12. User friendly interface

Design criteria require an intuitive user-friendly interface
for the powerful feature sets being offered. A graphical user
interface needs to be implemented that mimics the internal
workflow to facilitate user input and information retrieval. The
system automatically guides the users along the different steps
they need to go through in order to get the task completed. It
is desirable to avoid too complicated interfaces that may be
perceived as hostile for the final user.
13. Staff training
Modern LIMS are extremely sophisticated applications
with powerful capabilities. Unfortunately, most LIMS provid-
ers fail to communicate this clearly and without specialized
training and a qualified technical support, it is common that
many of the software capabilities remain unused. It is recom-
mended that there is informatics support (e.g., an informatics
engineer) capable of understanding, managing, and configur-
ing the LIMS as needed.
Acknowledgment
AGM and IB are supported by Obra Social Kutxa and ISCIII

(RD09/0076/00068).
References
1. Nakagawa AS (1994) LIMS: implementation 3. Olund G, Lindqvis P, Litton JE (2007) BIMS:
and management. The Royal Society of an information management system for
Chemistry, Cambridge http://trove.nla.gov. biobanking in the 21st century. IBM Systems
au/version/46681801 J 45(1):171–182
2. Litton JE (2011) Biobank informatics: con-
necting genotypes and phenotypes. Methods
Mol Biol 675:343–361
Chapter 5
Cryopreservation: Vitrification and Controlled Rate Cooling

Charles J. Hunt
Abstract
Cryopreservation is the application of low temperatures to preserve the structural and functional integrity of
cells and tissues. Conventional cooling protocols allow ice to form and solute concentrations to rise during the
cryopreservation process. The damage caused by the rise in solute concentration can be mitigated by the use
of compounds known as cryoprotectants. Such compounds protect cells from the consequences of slow cool-
ing injury, allowing them to be cooled at cooling rates which avoid the lethal effects of intracellular ice. An
alternative to conventional cooling is vitrification. Vitrification methods incorporate cryoprotectants at suffi-
ciently high concentrations to prevent ice crystallization so that the system forms an amorphous glass thus
avoiding the damaging effects caused by conventional slow cooling. However, vitrification too can impose
damaging consequences on cells as the cryoprotectant concentrations required to vitrify cells at lower cooling
rates are potentially, and often, harmful. While these concentrations can be lowered to nontoxic levels, if the
cells are ultra-rapidly cooled, the resulting metastable system can lead to damage through devitrification and
growth of ice during subsequent storage and rewarming if not appropriately handled.
The commercial and clinical application of stem cells requires robust and reproducible cryopreservation
protocols and appropriate long-term, low-temperature storage conditions to provide reliable master and
working cell banks. Though current Good Manufacturing Practice (cGMP) compliant methods for the
derivation and banking of clinical grade pluripotent stem cells exist and stem cell lines suitable for clinical
applications are available, current cryopreservation protocols, whether for vitrification or conventional slow
freezing, remain suboptimal. Apart from the resultant loss of valuable product that suboptimal cryopreser-
vation engenders, there is a danger that such processes will impose a selective pressure on the cells selecting
out a nonrepresentative, freeze-resistant subpopulation. Optimizing this process requires knowledge of the
fundamental processes that occur during the freezing of cellular systems, the mechanisms of damage and
methods for avoiding them. This chapter draws together the knowledge of cryopreservation gained in other
systems with the current state-of-the-art for embryonic and induced pluripotent stem cell preservation in an
attempt to provide the background for future attempts to optimize cryopreservation protocols.
Key words Embryonic stem cells, Induced pluripotent stem cells, Human, Cell line, Cryopreservation,
Vitrification, Slow cooling
1 Introduction
Since the first report of the successful derivation of a human

embryonic stem cell (hESC) lines by Thompson in 1998 and a
subsequent report on the reprogramming of human somatic cells
41
42 Charles J. Hunt
to form induced pluripotent stem cell (hiPSC) lines in 2007, there

has been considerable progress in optimizing cell culture condi-
tions for the maintenance of cells in the pluripotent state and their
controlled differentiation. Attention has also been given to the
translation of laboratory-based protocols to automated systems for
scale-up and scale-out to provide the large quantities of cells neces-
sary for in vitro drug and toxicity testing, as well as the modifica-
tion of cell culture methods and materials to provide cGMP
compliance for therapeutic applications [1–5].
For laboratory and commercial uses, which require the long-
term maintenance of viability and functionality, effective methods
of cryopreservation and low-temperature storage are a prerequi-
site. Such methods obviate the need to maintain cells in long-term
culture with the attendant problems of genetic drift, epigenetic
changes [6], and potential for contamination [7]. Cryopreservation
also allows the storage of master and working cell banks that cap-
ture and maintain a desired cell phenotype, thus making consis-
tent quality-controlled cells available and permitting the use of
functionally identical cells from well-characterized, contamina-
tion-free stocks.
From a commercial perspective, the availability of cryopre-
served material simplifies logistical considerations of transporting
cells within or between facilities and can provide a ready supply of
quality-controlled, freshly thawed cells for screening purposes
without the need for long-term continuous culture of the parent
cell line. Therapeutically, cryopreservation allows sufficient time
for comprehensive quality control and safety testing to be con-
ducted prior to the delivery of the cell therapy to the patient [8].
The progress made in optimizing culture conditions to main-
tain pluripotency of both hESCs and hiPSCs has so far not been
matched when considering robust and effective cryopreservation
procedures: protocols consistently return low functional recovery.
Though cryopreservation is a small part of the overall process in
the production of stem cells and their derivatives for research and
therapy, suboptimal cryopreservation can have a substantial effect,
not only in significantly reducing the numbers of cells available
post thaw, but also potentially by imposing a selective pressure,
during subsequent cell culture, through the survival of a nonrepre-
sentative, subpopulation of cells selected out by the preservation
process. Moreover, suboptimal cryopreservation may induce chro-
mosomal damage and epigenetic changes [9].
Two approaches have been taken to the cryopreservation of
pluripotent stem cells: slow cooling and vitrification [10]. To under-
stand the benefits and potential consequences of these different
approaches, an understanding of the basic principles underlying the
freezing and thawing of cells is required.
Cryopreservation: Vitrification and Controlled Rate Cooling 43
2 Principles of Cryopreservation
A comprehensive discussion of the effect of the physical and

biological response of cells and cellular systems to the application
of subzero temperatures is beyond the scope of this chapter and the
reader is referred to reviews by Mazur [11], Muldrew et al. [12],
and Pegg [13].
The biological effects of cooling are dominated by the freezing
of water to form ice: over 80% of the cell’s mass is water. Cooling
an aqueous solution below its equilibrium freezing point will at
some point induce water to form ice. Freezing is a nucleation-
induced event occurring by one of two processes: homogeneous or
heterogeneous ice nucleation [14]. The former occurs through the
random ordering of clusters of water molecules to form an ice
nucleus of sufficient thermodynamic stability to grow rather than
decay: homogeneous nucleation is a relatively unlikely event except
at temperatures near to −35 °C. Heterogeneous (or facilitated)
nucleation is catalyzed by a suitable solid or liquid interface in con-
tact with the solution that induces water molecules to form struc-
tures that promote ice crystallization. In practice, during
cryopreservation of cellular material, nucleation is induced at tem-
perature well above −30 °C and is heterogeneous in nature [15].
The probability of either type of event increases with both the
degree of cooling below the freezing point (referred to as super-
cooling or undercooling) and the volume of solution being cooled.
For cells in suspension, even at cell concentrations considerably
higher than those routinely cryopreserved for cell banking, cooling
below the equilibrium freezing point will result in nucleation in
the much larger extracellular compartment. The ice that forms is
essentially pure crystalline water containing virtually no dissolved
solute: the solutes, together with the cells, are concentrated in the
remaining liquid phase that will gradually decrease in volume as
temperature is lowered and freezing continues.
It is a basic tenet that a cell, in the absence of metabolic
processes, will adjust its internal concentration of water and per-
meating solute so that it is in equilibrium with the extracellular
compartment. This will lead cells, exposed to the changing tem-
perature environment, to respond by osmotic dehydration to the
osmotic disequilibria caused by the nucleation and growth of
extracellular ice and the resultant increase in external solute con-
centration. The kinetics of this response will depend on a number
of factors including the rate of cooling, permeability of the cell
membrane to water (Lp), and the cells surface/volume ratio. If the
rate of cooling is sufficiently slow, the cell will continue to reestab-
lish osmotic equilibrium through the movement of water out of
the cell and the intracellular compartment will remain free of ice.
If however the rate of change of temperature is rapid, or the cell’s
44 Charles J. Hunt
Fig. 1 Schematic of the response of cells to an imposed cooling rate. Once ice has formed in the extracellular
compartment, the cell will reestablish osmotic equilibrium by dehydration. If cooling rates are sufficiently slow,
cells will continue to dehydrate until all the freezable water in the system has been converted to ice. If cooling
rates are too high, or membrane permeability is insufficient to allow re-equilibration by water efflux, equilibrium
will be reestablished by intracellular freezing. At extremely high cooling rates the system can be made to vitrify
as the viscosity of the system suppresses all nucleation events and the solution forms a glass (not shown)
permeability or surface/volume ratio is insufficient to permit

compensatory water movement out of the cell, the intracellular
water will supercool below its nucleation point and the cell will
regain equilibrium with the external compartment by intracellular
freezing (Fig. 1).
The consequences of both these routes to osmotic equilibrium
can be damaging to the cell. The increase in solute concentration
reached is considerable; for instance, an isotonic saline solution will
freeze-concentrate 25-fold by −21 °C and damage to cells has
been linked to this rise in solute concentration [16, 17]. The phe-
nomenon of intracellular ice formation and its effect on cell sur-
vival were described by Mazur [18] who correlated the extent of
intracellular supercooling (caused by increasing cooling rates) and
the extent of intracellular ice formed, with cell survival. He dem-
onstrated a negative correlation between the predicted amount of
intracellular ice so formed and the survival of cells in suspension.
This was confirmed by Leibo et al. on studies with mouse ova [19].
It is important to note that while the fate of the population as a
whole is described by these studies, the fate of individual cells is
not. Within the cell population at intermediate cooling rates, there
will be a mixed response—some cells re-equilibrating by water loss
and others by intracellular ice formation [20].
An analysis of survival following freezing and thawing and the

influence of cooling rate led Mazur to formulate what became
known as the “Two-Factor Hypothesis” of freezing injury [21].
This theory has since underpinned all attempts to optimize cryo-
preservation procedures for cells, tissues, and organs.
2.1 The inverted “U” When plots of survival versus cooling rate are constructed, regard-
less of the assay method chosen to describe “viability,” cell survival
takes the form of an inverted U (Fig. 2). This has been demon-
strated for an ever-increasing variety of cell types. Damage, results
from two sets of opposing factors each operating at different cool-
ing rates: one set affecting survival at low cooling rates and the
other at high cooling rates. Two things are apparent from such
survival curves: first that an optimum cooling rate can be defined
for each cell type and second that the optimum cooling rate varies
over a broad range for different cell types. Two other factors not
evident from Fig. 2 also influence the two legs of the inverted
U. First, the rate at which the samples are re-warmed will tend to
have a differential effect on survival depending on the previously
applied cooling rate and second, the type and concentration of the
Fig. 2 Survival as a function of cooling rate. Percentage survival compared to nonfrozen controls for a range
of cell types. Data redrawn from: Mazur et al. - mouse bone marrow and human red blood cells (Ref. [22]);
Mazur - hamster oocytes (Ref. [23]); Hunt et al.—human cord blood cells (Ref. [24]). Cell survival shows charac-
teristic inverted U-shaped curves indicative of two opposing damaging factors: one operating at high cooling
rates, the other at low cooling rates
46 Charles J. Hunt
cryoprotectant (CPA) will also significantly influence survival.

In Fig. 2, the survival curves were generated by cooling and warm-
ing in the presence of CPAs. In the absence of a CPA absolute
survival is extremely low: for example, less than 2% survival was
reported for the mouse bone marrow cells shown in Fig. 2 when
frozen without cryoprotectant [22]. The effects of CPAs are also
asymmetric as their protective effect is exerted mainly on slowly
cooled cells and is proportional to their concentration [25].
2.2 Injury at High That cell survival correlates inversely with intracellular ice formation
Cooling Rates: (IIF) has been demonstrated experimentally in a number of studies
Intracellular Freezing [18, 19] and in most cases cells undergoing intracellular ice forma-
tion do not survive. The inability of a cell to regain equilibrium
through dehydration, either through lowered permeability to water,
a rapid lowering of temperature, or a combination of the two, will
cause cell water to supercool and nucleate. If the water permeability
of a cell is determined experimentally and the temperature coeffi-
cient of water permeability can be estimated, it is possible to deter-
mine the cooling rate at which intracellular ice formation is likely to
occur and the cooling rate adjusted to prevent its occurrence [26].
Numerous models have been developed to predict intracellular ice
formation [27, 28] which, though fundamentally differing in their
design, all give relatively similar results [29].
Cell membranes are efficient barriers to growth of ice and there
are few effective nucleators within the cytoplasm capable of nucle-
ating above −30 °C [30] leading to a debate on the mechanism
whereby intracellular nucleation of undercooled water at higher
subzero temperatures could take place. Three hypotheses predom-
inate, all of which assume a role for the plasma membrane in
IIF. The membrane pore hypothesis involves the seeding of intra-
cellular ice through preexisting aqueous pores in the membrane
[31]. In this hypothesis, the plasma membrane acts as an effective
barrier to ice nucleation above a certain temperature. Below this,
the radius of curvature of the developing ice dendrite is sufficiently
small to grow through or seed the supercooled intracellular water.
Studies by Acker et al. [32] with cell monolayers and Berger and
Uhrick [33] with tissues support such a proposition. Water chan-
nels (known as aquaporins) have been found in some, but by no
means all cell types, while theories of the diffusion of both water
and solute molecules through membranes postulate the transient
formation of cavities in the bilayer which could provide a route for
ice crystal propagation through the membrane [11].
Damage to the plasma membrane during freezing as a cause,
rather than a consequence, of intracellular ice has been proposed
based on experimental observations in plant protoplasts [34, 35]
where rupture of the protoplast was directly observed before IIF
occurred. In this hypothesis of osmotic poration [12], pores are
generated in the plasma membrane as a consequence of thermal
fluctuations during cooling. These may be expanded by the inter-

action of water with the pore edge during high water flux caused by
the rapidly increasing osmotic imbalance between the extracellular
and intracellular compartments. The pore will collapse once the
water flux is reduced but in the presence of extracellular ice the pore,
if of a suitable radius, will allow seeding of the supercooled intracel-
lular compartment by extracellular ice.
The third theory of intracellular nucleation proposes that the
plasma membrane does not need to be breached to catalyze intra-
cellular ice nucleation. Heterogeneous nucleation occurs as a result
of localized topographical changes to the plasma membrane occur-
ring as a result of the action of extracellular ice on components of
the membrane [28]. Here, extracellular ice plays an indirect, rather
than direct, role in IlF and the nature of this interaction, be it
chemical, mechanical, ionic, or electrical, is not known.
Whatever the mechanism of nucleation, the formation of intra-
cellular ice is generally considered to be lethal to cells in suspension
and to single attached cells [36]. The widely held view is that IIF
damages through mechanical disruption of intracellular membranes
with accretion of water molecules to the growing ice crystal and
redistribution of water from small ice crystals to larger ones. The
observed increase in post-thaw damage following slow re-warming,
in systems where IIF has occurred, is attributed to this recrystalliza-
tion phenomenon and has led to the adoption of rapid warming
techniques to maximize cell recovery.
Mechanical damage is by no means the only form of damage
attributed to IIF. Other mechanisms include thermal shock [37],
osmotic injury [34], protein denaturation [35], and gas bubble
formation [38].
That intracellular ice may be innocuous and even protective
under certain circumstances has also been postulated. The fact that
rapidly cooled cells can survive the presence of intracellular ice, if
rapidly warmed, suggests that intracellular ice per se is not invari-
ably lethal, rather the amount of ice, the ice crystal size [39], its
location [40], and the mechanism of formation [32] are the causes
of any cellular damage and the innocuous effect of intracellular ice,
if suitably controlled or minimized, has been reported for mamma-
lian embryos [41].
In a study, which may be of direct relevance to pluripotent
stem cells cryopreserved as adherent colonies or free floating
clumps, Acker and McGann [42] demonstrated a protective effect
of IIF in confluent monolayer cultures of MDCK and hamster
fibroblasts cells. Extracellular ice nucleation at −10 °C (which
resulted in ~100% IIF of adherent cells in the monolayer) resulted
in increased post-thaw survival, after subsequent cooling at 1 °C/min
to −40 °C, compared to nucleation at −5 °C, where only a fraction
of the cells showed IIF. Similar treatment of single cells in suspen-
sion showed no such protective effect. However, this is in direct
48 Charles J. Hunt
contrast to other reports for adherent cells, including human

mesenchymal stem cells (hMSCs), which indicate increased cell
damage from conditions that lead to IIF [43, 44].
2.3 Slow Intracellular ice can be avoided by cooling at rates that are low
Cooling Injury enough to allow sufficient water movement out of the cell. The cell
maintains chemical equilibrium with its surroundings throughout
cooling, until all freezable water has been removed. Although all
the ice formed in the system is extracellular, damage to the cell can
still take place as shown by the left-hand arm of the survival curves
in Fig. 2. Extracellular ice in dilute single-cell suspensions is prob-
ably innocuous and the mechanisms of damage are believed to
relate to the increased solute concentrations experienced by the
cell during the freezing process. This so-called solution effect
injury was first postulated by Lovelock [16] who demonstrated in
red cells that the hemolysis caused by freezing to a given subzero
temperature could be mimicked by exposing the cells to the same
concentration of solute (NaCl) that occurred at that subzero tem-
perature, but exposing them to it at 0 °C, before returning them
to isotonicity. This has been confirmed by others [45]. The asser-
tion that solute concentration alone is responsible for slow cooling
injury has been challenged. Damage has been attributed to the
consequences, direct or otherwise, of the growth of extracellular
ice and the sequestration of the cells in the diminishing unfrozen
solute channels within it [46], though the interpretation of the
evidence for the unfrozen fraction as a contributor to slow cooling
injury has been challenged [47].
Other mechanisms to explain the damage caused by slow cool-
ing invoke excessive cellular dehydration as the damaging mecha-
nism either as a consequence of shrinkage beyond a minimum
tolerated cell volume [48] or through membrane fusion events,
triggered by the shrinkage of the cell, with consequential post-
thaw damage [49]. However, evidence for a minimum tolerated
cell volume is lacking while that for membrane fusion events comes
almost entirely from plant protoplasts.
In studies on the influence of cell concentration (as a percent-
age of cells by volume) on the survival of cells after slow cooling,
high hematocrit (red cells) or high cytocrit (cultured cells) has been
shown to have an adverse effect on recovery of slowly cooled cells.
This too may be of relevance to pluripotent stem cells. Red cells
have been shown to exhibit increased hemolysis at hematocrits
above 50% [50] and slowly frozen hepatocytes also exhibit reduced
survival [51]. Kruuv [52] reported reduced survival after slow
freezing for V79 hamster cells when frozen as aggregates of ≤ 1000
cells compared to cells frozen as cell suspensions. The survival of
human bone marrow stem cells was reported to be reduced when
the cell concentration was increased from 1 × 107 to >2 × 108 cells/
ml under conditions where the volume fraction occupied by the
cells was high [53].
2.4 Influence Warming rate can have a profound influence on post-thaw survival
of Warming Rate when cells are cooled at high cooling rates (those on the right-
hand arm of the survival curves in Fig. 2). Here, rapid rewarming
substantially increases survival compared to rewarming slowly. This
is generally explained on the basis that rapid rewarming prevents or
minimizes ice recrystallization, thereby preventing small innocu-
ous intracellular ice crystals from growing larger and damaging the
cell; slow rewarming providing more time at higher subzero tem-
peratures for recrystallization to occur.
The influence of warming rate on slowly cooled cells is more
complex. While generally speaking, rapid rewarming confers simi-
lar benefits to those conferred on rapidly cooled cells, i.e., increased
survival when compared to slow rewarming, there are examples
where warming rate appears either to have little influence on the
survival of slowly cooled cells or where slow rewarming has been
shown to be beneficial [54, 55].
For slowly cooled cells where rewarming has been shown to
adversely affect survival, the cause is likely to be a recapitulation of
the damaging events described above for slow cooling; as the con-
ditions imposed on the cell during warming are in the main a
reversal of those during cooling. Damage incurred during freezing
is only likely to be expressed during thawing and, where such
events are time dependent, a slow freeze/slow thaw is likely to be
more damaging than one where the warming component is more
rapid.
2.5 The Optimum The survival curves for each cell type in Fig. 2 show an optimum
Cooling Rate cooling rate. This is explained by the interaction of two damaging
mechanism, solution effects and intracellular ice formation: the
former operating at slow cooling rates and the latter at rapid cool-
ing rates [21]. The terms “rapid” and “slow” are relative, relate to
the water permeability and surface/volume ratio of the cell type in
question, and are therefore different for different cell types. Slow
cooling may be defined as any rate of cooling below which the cell
can continue to respond to the rate of change of temperature, and
the consequential increase in extracellular ice, solely by water efflux
and cell shrinkage. Under these circumstances damage is attribut-
able to solution effects caused directly or indirectly by the elevated
solute concentration (Fig. 3). The effects of slow cooling injury
accumulate with increasing exposure time to damaging solute con-
centrations. Thus, increasing the cooling rate will result in shorter
exposure times and increased survival.
Rapid cooling may be defined as any rate of cooling above
which the cells fail to maintain chemical equilibrium by water
efflux, with equilibrium restored by the nucleation and growth of
intracellular ice. The consequences of intracellular ice formation
will depend on the warming rate but will generally lead to lethal
events and reduced survival. Survival will continue to be reduced
as cooling rates continue to increase. The optimum cooling rate is
50 Charles J. Hunt
Fig. 3 Schematic showing the mechanism of injury. At slow cooling rates, solution effects predominate with long
exposure time leading to high levels of damage. This is mitigated by increasing the cooling rate. Increasing the
cooling rate will lead to an increase in the incidence of intracellular ice and a consequential reduction in survival of
cells on thawing. The optimum cooling rate results from the minimization of these two opposing factors
thus a compromise between these two competing factors; when

cooling is rapid enough to reduce damage from solute effects but
slow enough to reduce the incidence if IIF in the cell population.
2.6 Multistep The optimal cooling rate avoids significant IIF while minimizing
Cooling and Controlled the time of exposure to high salt concentration. Such procedures
Nucleation of Ice imply a constant cooling velocity over the temperature range at
which the damaging events occur (generally considered to be from
the freezing point to about −40 °C) with controlled rate cooling
machines or passive cooling devices providing linear (or near linear)
cooling rates over this temperature range.
An alternative strategy to minimize damage is two-step cooling
[34]. Here, cells are cooled rapidly to a constant subzero tempera-
ture and then held at that temperature to allow sufficient water
efflux to prevent IIF during subsequent rapid cooling to the
storage temperature; reducing exposure time to damaging solute
concentration during this final phase. The introduction of a hold
period at high subzero temperatures (−5 °C to−15 °C) has also
been advocated to allow controlled ice nucleation. The effects of
controlled nucleation on cell survival have recently been reviewed [56].
Controlled nucleation has been demonstrated to improve the
survival of slowly cooled hESCs and multipotent mesenchymal
stromal cells [57] using conventional seeding methods [58, 59].
Chemical ice nucleants such as cholesterol have been employed
for single-cell suspensions [60] but may be of limited utility for
cells requiring GMP compliance for clinical applications. However,

new GMP-compliant materials are being developed and have been
shown to provide improved recovery of hESCs after slow cooling
(Morris J and Bruce K: personal communication). Recently, a multistep
cooling method has been advocated for hiPSCs [61].
3 Cryopreservation
The discovery that glycerol could protect fowl spermatozoa from

the effects of freezing [62] and subsequently that the compound
dimethyl sulphoxide [63] had a similar effect on red cells, has led
to their widespread use as cryoprotectants, along with a wide range
of other compounds such as ethylene glycol (EG), propylene glycol
(PG), hydroxyethylstarch (HES), and polyvinyl pyrollidone (PVP).
All these compounds have the same effect—that of mitigating the
damaging effects of freezing. However, the protection conferred
by them is not equal across all cooling rates.
Figure 4 shows the effect of increasing glycerol concentration
on the survival of mouse marrow stem cells as a factor of cooling
Fig. 4 The moderating effect of cryoprotectants. Survival of mouse marrow stem cells as a function of cooling
rate. The cells were suspended in Tyrode’s solution containing the stated concentration of glycerol and cooled
over a range of cooling rates. Warming was rapid. Redrawn from Ref. [22]. Glycerol increases survival predomi-
nantly at slow cooling rates, with little effect at supraoptimal (rapid) cooling rates. Increasing glycerol concentra-
tion also reduced the optimal cooling rate from approx. 100 °C/min at 0.4 M to approx. 2 °C/min at 1.25 M
52 Charles J. Hunt
rate [22]. In the absence of a CPA less than 2% of cells survive

across all cooling rates. As glycerol concentration increases, survival
increases. However, the relative increase in survival is greatest at
slow cooling rates (approximately a ninefold increase in survival at
the minimum cooling rate compared to a less than 1.5-fold increase
at the maximum cooling rate) resulting in a lower optimal cooling
rate. The lack of a protective effect at rapid cooling rates shows
that in general cryoprotectants have little effect on mitigating the
damage caused by IIF; their main protective effect being exerted
against solution effect injury.
3.1 The Mechanism Glycerol and DMSO, together with EG and PG, fall into a class of
of Action CPAs known as permeating cryoprotectants that are generally
of Cryoprotectants small nonionic molecules with high solubility in water that can
diffuse freely through the plasma membrane and equilibrate in the
intracellular compartment. Their mode of action was described by
Lovelock [64] on a colligative basis. In a partially frozen solution
the total concentration of solutes in the unfrozen fraction is fixed
and independent of the nature of the solutes. As the damaging
effect of salts is directly related to concentration, lowering that
concentration, by replacing a portion of the damaging solutes in
the partially frozen solution with a CPA, will result in less damage
at any given subzero temperature. By lowering the amount of
ice formed as well as acting as a secondary solvent for salts [65],
the CPA will cause the cell to be exposed to a lower salt concentra-
tion at say −15 °C than would be the case in the absence of the
CPA. The damaging effect of salts is related not only to concentra-
tion but also to the temperature of exposure. Thus, put another
way, in the presence of a CPA the cell will be exposed to the same
damaging salt concentration but exposure will occur at a lower
subzero temperature where its damaging effect is reduced.
However, during the freezing process, salts are not the only
solutes to be concentrated—the CPA undergoes a similar freeze
concentration and has been shown to contribute to the hemolysis
of frozen thawed red cells [45]. Thus, protection comes at a price
in that concentrations of CPA that overall have a beneficial pro-
tective effect can also contribute to “solution effect” injury at
slow cooling rates.
While the evidence for this mode of action is strong, other
mechanisms of action have been proposed including stabilization
of the plasma membrane by the CPA [66]. Such interactions have
been proposed for nonpermeating cryoprotectants. Nonpermeating
CPAs, such as PVP and HES, are generally large molecular weight,
long chain polymers that do not cross the plasma membrane and
remain in the extracellular compartment during freezing. Their
mode of action is the matter of debate but has been attributed to
both a colligative effect and the prefreeze dehydration of the cell;
reducing intracellular water and increasing the likelihood of the
cell maintaining equilibrium by water loss rather than by intracel-

lular freezing. The high viscosity of compounds such as HES may
also act to inhibit ice nucleation during cooling and recrystalliza-
tion during warming [67]. HES in particular has been shown to be
effective, in combination with reduced concentrations of DMSO,
in the cryopreservation of hESCs by slow cooling [68].
3.2 Damage Caused Though protective, CPAs can themselves be the cause of cell dam-
by Cryoprotectants age induced by inappropriate introduction or removal, as well as by
their intrinsic toxicity. The permeability of the plasma membrane
to permeating CPAs is many-fold lower than its permeability to
water and CPAs thus exert a transient osmotic effect on the cell.
During addition, water will leave the cell in response to the initial
osmotic disequilibrium before re-swelling as the CPA (along with
water) permeates the cell. The magnitude of the cellular volume
excursion will depend on the permeability to CPA and water, the
initial CPA concentration, and the temperature of exposure.
Exposure to nonpermeating CPAs will also cause shrinkage. However,
the cells will remain shrunken until the nonpermeating CPA is
removed or diluted; in which case the extent of shrinkage will
depend only on the CPA concentration and the water permeability
of the cell.
The reverse is true during the removal of CPAs. Post-thaw elu-
tion protocols in which the external CPA concentration is rapidly
lowered (by, for example, centrifugation and resuspension of the
cell in CPA-free medium in a single step) can lead to excessive
swelling of the cell resulting in damage and cell lysis. The need to
adjust elution (and addition) protocols to ensure that the cell vol-
ume excursion is tolerated by the cell and osmotically induced
damage is avoided, has been demonstrated for cord blood and
other cells [24, 69, 70]. Approaches to designing effective addi-
tion and elution procedures are available [69]. One such approach
employs an “osmotic buffer”—a nonpermeating solute such as
sucrose or dextran that when added to the elution solution can
restrict the extent of cell swelling [71].
Clinically, DMSO has been shown to produce allergic reactions
in patients infused directly with hematopoietic stem cells containing
the CPA and has also been shown to induce apoptosis in the devel-
oping central nervous system of mice [72]. Many centers have there-
fore perfected protocols to elute DMSO before reinfusion of stem
cells into recipients, particularly children. Cells too can be affected
by molar concentrations of CPA normally used to protect the cells
from freezing and thawing. With hES and other cells in vitro, DMSO
is known to be a powerful inducer of differentiation [73] and can
trigger apoptosis [74] when used in low concentration (ca.1%) over
extended periods of time (days) at elevated temperatures (+37 °C).
It has also been shown to have an effect on the epigenetic profile of
murine stem cells [75].
54 Charles J. Hunt
Chemical toxicity is time-, temperature-, and concentration

dependent. Toxicity varies from cell type to cell type and the
accepted practice has generally been to introduce and remove the
cryoprotectant at lower temperatures (+4 °C). Chemical toxicity is
not restricted to DMSO. A recent study investigating the effect of
four CPAs (DMSO, EG, PG, and glycerol) on recovery of hiPSCs
showed that all four CPAs were damaging when cells were exposed
to the CPA at 37 °C [61]. Damage was less severe with PG and EG
than with DMSO, while glycerol which showed the least toxicity
produced the worst recovery after freezing and thawing (due prob-
ably to osmotic damage occurring during either addition or
removal). The effect of exposure at ambient temperature was not
explored in this study, but hematopoietic stem cells have been
shown to tolerate exposure to DMSO at ambient temperatures for
periods of time consistent with those necessary for all prefreeze cell
manipulations to be carried out [24, 76].
While the concentrations employed to cryopreserve cells by
conventional freezing (in the range of 0.5–2 M) are generally well
tolerated by cells, providing exposure times and temperatures are
restricted to those sufficient to allow equilibration of the cell with
the CPA, the far higher concentrations employed to achieve vitrifi-
cation can be severely toxic and damaging osmotically, necessitat-
ing different approaches to their introduction and removal.
4 Vitrification
Vitrification is the solidification of a solution without crystalliza-

tion and the growth of ice. This is achieved when solutes in the
system are sufficiently concentrated, or the system cooled suffi-
ciently rapidly, that the increased viscosity inhibits nucleation and
prevents the growth of ice. As cooling continues, viscosity continues
to increase until all molecular motion is (for all practical purposes)
halted and the solution becomes a glass. In this condition, the sys-
tem displays the properties of a solid but retains the molecular
structure of a liquid [13].
Even during slow cooling, some vitreous material will be
formed in the system as it cools. If cooling is sufficiently slow that
all water in the system (including cellular water) is removed to
form extracellular ice, then the resulting concentrated solute phase
including the cells embedded in it will form a glass. Both solutes
and CPA will concentrate with, for instance, a 10%w/w glycerol
solution reaching a concentration of approximately 67% w/w by
this point. Thus, while a vitreous component to the system exists,
it is confined to the channels between extracellular ice crystals.
Such a system cannot be considered to be truly vitrified. To achieve
true vitrification of the entire system using, for instance, glycerol
would require the addition of a 67%w/w solution before cooling
commenced. Such a concentration would be highly toxic to the

cells and its addition and removal would produce significant
osmotic stress.
Nevertheless, if the issues of toxicity and osmotic damage can
be overcome, vitrification offers some potential benefits.
Vitrification avoids the formation of intracellular ice with its atten-
dant damaging effects. The avoidance of extracellular ice in the
system also means the avoidance of “solution effect” injury, since
freeze-concentration of damaging solutes is avoided. Moreover,
during the cooling phase, cells are exposed to less concentrated
solutions of the cryoprotectant for shorter periods of time than
would be the case in conventional freezing [77]. Thus, cooling and
rewarming at an optimum rate is unnecessary; only a
cooling/warming rate sufficient to avoid crystallization is required.
This foregoes the need for expensive and complex equipment [78].
Vitrification may be achieved in one of two ways: by increasing
the concentration of the CPA to levels sufficient to avoid ice for-
mation whatever the imposed cooling rate—equilibrium vitrifica-
tion, or by employing extremely high cooling rates with the
requirement for lower (and therefore less toxic) concentrations of
the CPA—nonequilibrium vitrification.
4.1 The Equilibrium This approach requires high (and potentially toxic) multimolar
Approach concentrations of CPA. Chemical toxicity is time, temperature,
and concentration dependent and this approach to vitrification has
generally required both the careful formulation of cryoprotectant
mixtures, in order to lower the toxicity of each individual compo-
nent of the CPA, and their introduction in a stepwise fashion at
increasingly lower temperatures. However, osmotic damage is
increased by reduction in temperature and thus any protocol for
vitrification using this approach is often a compromise between
inflicting chemical toxicity or osmotic damage on the cell. Although
effective protocols for the introduction and removal of high con-
centrations of CPA at room temperature have been developed,
exposure times and concentrations are critical [79]. Another
approach, first developed by Farrent and others [80, 81], exposes
the cells to increasing concentrations of CPA but does so at pro-
gressively lower subzero temperatures such that the system remains
above its equilibrium freezing point at each subzero exposure tem-
perature. This approach to equilibrium vitrification, known as the
liquidus-tracking method, as it tracks the liquidus or equilibrium
freezing point curve of the system, has been applied to the preserva-
tion of articular cartilage, a previously poorly cryopreserved tissue,
with significant success [82].
4.2 Nonequilibrium While the above approach allows the vitrification of cells at slow
Vitrification cooling rates, nonequilibrium vitrification takes advantage of the
fact that, in the presence of much lower concentration of
56 Charles J. Hunt
cryoprotectant, both nucleation and ice crystal growth can be pre-

vented by employing extremely rapid cooling rates [13]. However,
the vitrified state attained is metastable and to ensure the system
remains ice free, warming rate becomes of crucial importance as the
system will traverse the zones of ice nucleation and ice crystal growth
during re-warming. During this phase, devitrification (freezing and
ice crystal growth) will take place if warming is too slow.
The choice of cryoprotectant(s) under these conditions is also
important since not only are the critical cooling and warming rates
necessary to prevent ice formation different for different CPAs but
the degree of toxicity (used in its broadest sense to include all forms
of CPA-related damage) will be ranked differently for different cell
types. The concentration of CPA required for vitrification can be
reduced by the addition of other solutes and glass-promoting agents
that act to lower the critical cooling and warming rates necessary to
promote vitrification. Using these extracellular additives, various
combinations of CPA (notably EG and DMSO) have been applied
successfully to many cells and tissues including embryos, cord blood,
and amnion-derived mesenchymal stem cells [78, 83, 84].
4.3 Vitrification Nonequilibrium vitrification, using a protocol based on that used for
of Embryonic Stem the cryopreservation of bovine embryos, has seen widespread accep-
Cells tance for the cryopreservation of hESCs (see Ref. 85 for detailed pro-
tocol). The adoption of this method rests largely on studies by three
groups, all of which reported recovery (and expansion of undifferen-
tiated colonies) of >75% compared to <25% for samples cooled using
a standard slow cooling protocol [86–88]. In contrast to mouse
embryonic stem cells, these and other studies also indicated that
dissociation of the colonially-grown hES cell into a single cell suspen-
sion for cryopreservation (or passage) tended to lead to differentia-
tion and cell death and that the optimal size of the cell cluster for
vitrification was of the order of 100–500 cells [89].
Briefly, this protocol requires the stepwise exposure of hESC
colony fragments to two vitrification solutions of increasing cryopro-
tectant concentration, the common components of which are DMSO
and EG. The composition of the vehicle solution varies, with differ-
ences in sucrose concentration, the presence or absence of serum and
the buffer used. Colony fragments are exposed to the two vitrifica-
tion solutions sequentially: VS1 containing 10% v/v each of DMSO
and EG and VS2 containing 20% v/v each of DMSO and EG with
added sucrose. Exposure to the vitrification solutions is brief
(60 s and 25 s respectively at either room temperature or 37 °C).
EG has been shown to be less toxic to hESC than DMSO
[61]. Using mixtures of cryoprotectants in lower concentrations
helps to reduce the intrinsic toxicity of each, while the two-step
addition protocol will reduce osmotic transients. Exposure times
are brief and unlikely to allow full equilibration of the CPA with
the cells. This and the use of sucrose, a nonpermeating CPA, will
preshrink the cells reducing the likelihood of IIF during the

cooling process.
To achieve vitrification using these solutions, very rapid cooling
rates are required, This is achieved by the direct immersion in LN2
of finely drawn capillaries holding ultra-small volumes (~20 μl or
less) of the vitrification solution containing the colony fragments.
This method, first introduced for bovine embryos, has been termed
the OPS method (for open pulled straw).
To avoid ice crystallization during thawing the straws are
re-warmed as rapidly as possible by direct immersion of the tip of
the loaded straw into pre-warmed culture medium containing
sucrose. Once thawed the colony fragments are then expelled into
this medium and transferred stepwise through cryoprotectant
wash-out solutions, containing decreasing concentration of sucrose
(here acting as an osmotic buffer), until they are plated into culture
medium. An alternative method with direct exposure to growth
medium without stepwise elution of the cryoprotectants has also
been used with no noticeable deleterious effects [85].
The ultra-small volumes within each straw and the need to
maintain the sample below the glass transition temperature to
avoid devitrification during storage and transportation make this
method technically challenging even in a laboratory setting and are
likely to constrain its use commercially. The direct immersion of
the open straw in LN2 to attain the rapid cooling rates necessary to
achieve vitrification is also likely to prove problematic, particularly
in a clinical setting where the use of open straws would markedly
increase the possibility of the contamination of the vitrified material
by adventitious agents.
The process of preparing and vitrifying HESCs in individual
straws by the OPS method is time consuming and the short exposure
times employed are likely to lead to an inconsistent and operator-
dependent product. At best, this method permits the production
of small laboratory-scale banks of nonhomogeneous material and
methods that allow scale-up will still be required if vitrification is to
provide a viable option in a commercial setting.
Methods have been proposed for the scale-up of this process.
These involve “bulk” vitrification of partially dissociated colonies
in cell strainers [90] or modified cryovials [91]. Using these meth-
ods the equivalent of a 35 mm culture dish (approx. 100–150 cell
clumps) could be transferred to a single cell strainer (equivalent to
10–20 straws) in a procedure taking not more than 5 min. When
compared to hES cells recovered by the OPS method, no signifi-
cant differences were detected in either the rate of reattachment,
the degree of differentiation (measured at day 7) or in pluripotency
of the surviving cells.
Surface-based vitrification techniques have also been developed
that allow modest scale-up. Heng et al. [92] originally proposed a
design for a culture plate with detachable wells in which whole
58 Charles J. Hunt
adherent colonies could be vitrified. Though not taken up per se,

methods utilizing this approach with Thermanox© cultivation discs
[93] and whole plastic culture plates [94] have produced recoveries
equivalent to the standard suspension-based vitrification method.
However, all these methods still require direct immersion of the
cells in the cryogen. While methods and equipment exist for the
sterile filtration of LN2 [95], their applicability to cell banking and
the sterility of the coolant at the point of use is questionable. Since
none of the methods currently developed for “bulk” vitrification
employ closed systems, the potential for contamination directly
from the LN2 during vitrification and storage [96, 97], from other
cells [98], or from their containers is likely to prove a regulatory
barrier to clinical application, as well as providing a ready-source
for the spread of laboratory-acquired contamination.
Solid surface vitrification (SSV) avoids contact between the
cryogen and the sample by cooling a sterile solid surface on which
microdroplets can be vitrified [99]. This method has been com-
mercialized and used to vitrify murine ovarian tissue and porcine
blastocysts [100, 101] though its applicability for high through-
puts is questionable.
Separation of the cryogen from the sample has also been
achieved using methods developed for embryo and gamete cryo-
preservation. Richards et al. [88] used FDA-approved sterile 250 μl
embryo straws that could be sealed using a conventional heat
sealer. The results were comparable to the OPS method when
comparable volumes (i.e., 20 μl) of vitrification solution were used,
even though an order of magnitude difference in cooling rate
could be expected [102]. While the sealed straw prevents direct
contact between the sample and the cryogen, the external surface
of the straw may still be contaminated during handling and storage
with subsequent contamination of the thawing solution.
Disinfection of the surface prior to thawing is difficult due to the
potential for devitrification of the contents and a method that pro-
tects the straw from contamination is required.
The straw-in-straw method has been successfully employed for
mouse embryos [103] and neurospheres [104] and has been advo-
cated for human hESCs though a further reduction in the cooling
(and subsequent rewarming) rate, again by an order of magnitude,
is likely [78]. A modification to the straw-in-straw method employ-
ing a notched capillary tube within a sealed straw has recently been
employed for vitrification of the inner cell mass (ICM) of the
blastocyst. The preservation of the ICM, as an alternative to pres-
ervation of an ICM-derived embryonic stem cell line, offers an
alternative avenue for banking [105].
The use of cryovials for vitrification has also been reported
[106]. Nishigaki et al. successfully cryopreserved primate and
human embryonic stem cells in cryovials in a vitrification solution
without DMSO, replacing it with elevated EG concentrations and
polyethylene glycol (PEG). Plunge cooling rates for 200 μl volumes

were measured at ~125 °C/min and DSC measurements on the
chosen solution (containing 40% EG, 10% PEG, and 20% KSR)
indicated that the solutions would vitrify at this cooling rate. A
reattachment index was used to assess post-warming recovery after
1 day in culture and they obtained recovery rates of around 20% by
this method with surviving colonies expressing typical pluripotency
markers and the ability to form teratomas. Other studies [107]
using the same vitrification solution on hiPSCs (in which KSR was
replaced by Eurocollins solution) have confirmed the utility of this
solution when compared to a commercial alternative containing
DMSO. Recovery rates were slightly higher than previously
reported but in neither study were the exposure times, temperatures,
or method of addition/removal reported in detail and further
improvements in post-thaw recovery may well be possible. At the
UK Stem Cell Bank, we have also successfully vitrified colony frag-
ments from a range of embryonic stem cell lines in cryovials using
the VS1/VS2 vitrification solution described above and sample
volumes equivalent to that of the OPS method. Post-thaw recov-
eries were as good as or better than those for the OPS method
(L Young, personal communication).
Though methods such as those above help address the problem
of potential contamination and improve throughput, none really
provides a solution to scale-up or automation. At best, the sealed
straw and straw-in-straw methods may provide a means, acceptable
to the regulatory authorities, for small-scale cell banking of seed
stocks of stems cells for therapeutic application. For these and for
other logistical reasons the commercial and clinical applications are
most likely to involve conventional slow cooling methodologies
despite their perceived problems.
5 Cryopreservation of Pluripotent Stem Cells by Conventional Slow Cooling
Though a quality-by-design approach has been advocated for opti-

mizing cryopreservation processes [108], most studies on cryopreser-
vation of stem cells using conventional slow cooling, like those for
vitrification, have been largely empirical though a methodological
approach has been applied to optimizing cryopreservation proto-
cols for hematopoietic stem cells [109]. In this approach the bio-
physical parameters (Lp, Ps, and others) involved in the kinetics of
water loss (and thus cryopreservation injury in its broadest sense)
are obtained and used to model appropriate addition/elution pro-
tocols for the CPA. This in turn allows CPA toxicity and optimum
cooling rate to be determined free from the confounding effects of
other potentially damaging factors. This approach has also been
applied to murine ES cells [110, 111] and to hESCs [112]. In this
study, Xu et al. determined the membrane permeability of an hESC
60 Charles J. Hunt
line (H9) and a commercially available hiPSC line (SiDSH) to

water and CPAs, including DMSO and PG. The results showed
biophysical differences between the hESC and hiPSC lines and,
compared to murine ES cells, the human cells were more perme-
able. Modeling, based on the data for DMSO and PG, indicated
that, though the biophysical parameters differed between the cell
lines, this did not translate into a significant difference in predicted
optimal cooling rate. However, when cells were compared against
a standard 1 °C/min cooling rate, those frozen at the calculated
optimal cooling rates did not show any improvement in immediate
post-thaw survival. While this type of data is fundamental to estab-
lishing optimal cooling rates, further studies are required as assess-
ing immediate post-thaw survival alone is unlikely to be a
satisfactory marker for long-term function (J Man personal com-
munication). This highlights the difficulty in choosing appropriate
assays to measure not only immediate post-thaw recovery in cell
clumps of varying and undefined size, but also the ability of these
agglomerates to reattach, proliferate, and maintain pluripotency
for extended periods in culture and the n-points at which these
assays should be applied. Comparative studies are thus difficult to
interpret and the importance placed on evidence of pluripotency
and karyotypic stability, as primary indicators of the effectiveness of
the cryopreservation procedure, has perhaps been overemphasized
as such tests are generally carried out a number of days or weeks
post-thaw when the effects of lethal and sublethal injury will have
been diluted out. Despite this, a number of studies have investi-
gated isolated cryobiological variables though the results produced
are often conflicting.
Many of the early studies by Reubinoff and others [86–88, 113,
114] using slow cooling with a variety of cryoprotectants reported
low recovery and increased differentiation following thawing. The
protocols used were adapted from those used to cryopreserve
murine ES cells or mammalian cell lines with reported success.
However, Kashuba Benson has reported that there is considerable
cell line to cell line variation in the recovery of murine ES cells fol-
lowing slow cooling; with post-thaw recoveries ranging from 10 to
90% [110]; not at all dissimilar to the ranges reported across the
literature for hESCs where some studies have reported relatively
high survival [59, 115–117].
5.1 Influence There has been little research to determine the optimum slow
of Cooling Rate cooling rate. The early hES studies, comparing slow cooling and
vitrification, used slow cooling rates ranging from 0.5 °C/min to
4 °C/min [68, 87, 113, 118]. Kushuba et al. have recently reported
an optimized protocol for mouse ES cells which utilizes a 1 °C/min
cooling rate [119], though this was not optimal for all the cell lines
studied.
As discussed above, calculated optimum cooling rates for one

hES cell line (H9) have been reported to be of the order of 10 °C/
min [112]; though immediate post-thaw recovery was comparable
to cells frozen at 1 °C/min. In another recent study, employing
two-step cooling and a range of post-thaw assays, optimum recov-
ery for cells in a DMSO/HES/serum replacement medium was
obtained by cooling at 0.4 °C/min to −40 °C followed by 5 °C/
min from −40 °C to −90 °C [120].
5.2 Passive Cooling The choice of equipment to deliver the chosen cooling rate rests
Devices or Controlled between passive cooling devices (PCDs) and controlled rate freez-
Rate Freezers ers (CRFs). PCDs, in which cooling is achieved using an external
cold source (often a −80 °C freezer) and an insulated container to
hold the samples, are the simplest and cheapest solution. Varying
the insulating material and the temperature of the cold source will
allow some control of the cooling rate and the temperature range
over which cooling may be considered to be reasonably linear.
However, cooling is asymptotic and cooling rate will become much
slower as the end point temperature is approached.
Commercially available PCDs, in which the conducting
medium is isopropanol (Mr Frosty, Nalgene) or a high-density
insulating foam incorporating a heat sink device (CoolCell,
BioCision), have been designed to offer a cooling rate of approxi-
mately 1 °C/min between about −10 °C and −40 °C. However,
nucleation is uncontrolled without the addition to the CPA of an
ice nucleating agent and cooling at rates other than that for which
the PCD was designed is not easily achievable.
True linear cooling, controlled cooling at rates away from
1 °C/min, multistep cooling, and controlled nucleation are only
achieved using CRFs. The coolant utilized by CRFs is LN2 that is
dispersed in bursts into the sample chamber under pressure through
a solenoid valve. The CRFs are usually programmable and control
the rate of change of temperature through activating the solenoid
valve in conjunction with a heating coil to provide precise tempera-
ture control. A recent novel CRF (Cell Alive System, ABI Corp.,
Japan) containing an oscillating magnetic field has been used to
cryopreserve hESCs [121]. The field promotes vibration of water
molecules during the freezing process to suppress ice crystal growth.
Most LN2-cooled CRFs will provide cooling to temperatures
below −100 °C over a wide range of cooling rates up to about
30 °C/min. An alternative to the LN2-cooled CFR are those that
operate on the Stirling Cycle principle (Viafreeze, Asymptote Ltd.,
UK) using electrically driven heat pumps (Stirling Cycle engines)
which obviate the need for LN2 and may thus be readily used in
a cleanroom environment. These have been used to cryopre-
serve hESCs in 50 vial batch sizes or in straws [122]. Larger
versions with a 250 vial or 100 ml blood bag capacity are also
available [123]. Unlike LN2-cooled CRFs, those powered by
62 Charles J. Hunt
Sterling Cycle engines are restricted on a practical basis to cooling

rates below 2 °C/min.
5.3 Controlled As previously discussed, the control of the compartment in which

Nucleation of Ice ice nucleation occurs has been shown to be important for many
cells and tissues and may be so for hESCs frozen as an agglomeration
of cells requiring maintenance of cell-to-cell contact.
The beneficial effect of seeding on the survival of a range of
hES cell lines after thawing has been demonstrated in a number of
studies. In a study by Ware et al. [58], survival was calculated as
combined colony number-colony size, relative to a prefreeze con-
trol. Clusters were frozen by controlled rate cooling in 10% DMSO
at cooling rates between 0.3 °C and 3 °C/min. High survival
(~80%) was obtained at cooling rates below 1.8 °C/min, but only
if the samples were seeded. The effect of seeding temperature on
survival was not fully investigated: however, seeding at −7 °C and
−10 °C produced similar results. Yang et al. reported similar results
with seeding at −10 °C and a cooling rate of 0.5 °C/min [59]. Li
et al. [115] obtained reattachment and recovery rates of over 50%
using programmed cooling (1 °C/min) with seeding at −7 °C
after single-step addition of the CPA (10% DMSO) with compara-
tively low survival in the group cryopreserved without seeding. A
similar study including two-step equilibration of the CPA also
demonstrated improved recovery after seeding at −7 °C (>50%) as
well as highlighting another possible confounding factor: variabil-
ity in the cell line response to slow cooling injury [116]—as already
reported for murine ES cells.
The effectiveness of controlled nucleation in cases where the
cells are frozen as a clump may be related to a modification in the
distribution of ice. It has been demonstrated that intracellular ice
can be nucleated cell-to-cell via gap junctions [32, 43]. The pres-
ence of functional gap junctions has been demonstrated in hES
cells [124] and gap junction communication has been implicated
in a number of cellular processes including cell proliferation, dif-
ferentiation, and apoptosis [125, 126]. Damage to the hESC clus-
ters caused by intercellular ice propagation, either by random
nucleation events within the cluster and propagation through the
gap junctions [127] or from surface catalyzed nucleation at its
periphery [128], followed by cell-to-cell propagation could lead to
disruption of the cell cluster affecting cell proliferation,
differentiation, and apoptosis on thawing [33]. Methods that
attempt to control the nucleation event, such as seeding, may
therefore act to initiate ice formation outside the cellular (or cell
cluster) compartment.
Multistep cooling programmes with or without seeding have
also been reported to improve recovery compared to either single-
step cooling protocols [129] or the nonlinear cooling profile pro-
duced by passive cooling devices [61]. The multistep cooling
profile developed by Katkov et al. [61] has also been applied

successfully to adherent colonies that have so far proved difficult
to cryopreserve by slow cooling unless encapsulation techniques
are employed.
Modification to the environment in which the cells experience
the freezing process also appears to modify the extent of cellular
injury, perhaps through preferential formation of ice in the encap-
sulating matrix. Ji et al. compared whole colonies frozen in suspen-
sion with colonies frozen while still adherent to the culture surface
with only a thin layer of cryoprotectant covering the cell layer
[130]. Survival was low but could be significantly improved by
embedding the adherent colonies within a layer of Matrigel prior
to slow cooling. A similar improvement in survival was reported
for human ESCs grown on microcarriers and encapsulated in algi-
nate [131, 132] and for murine ESC encapsulated in an RGDS-
functionalized alginate [133].
5.4 Effect The choice and mode of application of the CPA can have a signifi-
of Cryoprotectant cant effect on the recovery of many cell types including hESCs.
Commonly DMSO has been the CPA of choice used either singly
or in combination with a range of other CPAs. When used singly it
has been employed generally at a concentration of 10% w/v
(~1.3 M) often with a single addition and elution step. Two-step
or multistep addition and elution protocols when employed have
been shown to increase recovery due presumably to the avoidance
of CPA-induced osmotic damage. Valbuena et al. [134] investi-
gated the effect of stepwise addition of 2 M DMSO in a four-step
addition process at room temperature on hES cells. Though imme-
diate post-thaw survival was low, it was more effective than single-
step protocols and comparable to vitrified samples. More recent
studies have emphasized the beneficial effects of stepwise equilibra-
tion of the CPA often in combination with seeding during slow
cooling. Lee et al. demonstrated improved survival compared to a
single-step protocol when the two-step equilibration process was
employed [116] and stepwise elution of CPA from cryopreserved
adherent hiPSC has also been shown to improve survival [61]
though not in all cases [135].
Multistep addition and elution protocols will reduce the dam-
aging effect of osmotic transients but increase the exposure time to
the cryoprotectant, potentially exacerbating intrinsic chemical tox-
icity. The toxic effect of CPAs has been demonstrated for hiPSCs
when equilibrated at 37 °C [61]. Reducing the exposure tempera-
ture will reduce toxicity as demonstrated for hematopoietic stem
cells [24]. Reducing the DMSO concentration alone has also been
shown to be beneficial for a range of human and nonhuman stem
cells while still conferring adequate protection [136–138].
Alternatively, the CPA formulation may be adjusted, to lower tox-
icity and reduce apoptosis [139]. Xu et al. reduced the DMSO
64 Charles J. Hunt
concentration to 7.5% w/v and incorporated 2.5% w/v PEG into the
CPA, producing improved recovery [140]. The use of 5% DMSO
in combination with 5% HES [66] or 6%HES/5%EG [141] and a
similar mixture of DMSO/HES with added plant hydrolysates
[68, 142] also produced improved recovery.
Trehalose, a nonpermeating disaccharide, has been used in
combination with DMSO for the cryopreservation of both fish and
human embryonic stem cells [143, 144] and with PEG and DMSO
for the cryopreservation of hMSCs [145]. Trehalose does not
readily penetrate cells, with only low concentrations (<0.5%) being
absorbed by passive diffusion or active endocytotic mechanisms
and in none of the cases referenced above were exposure times suf-
ficient, nor active methods used, to load this compound into the
cells. This suggests that trehalose was acting here as an extracellular
cryoprotectant. Its effectiveness at 0.1 M to 0.2 M when used in
combination with 5% DMSO has also been demonstrated for the
cryopreservation of murine embryoid bodies [146].
Alternatives to DMSO have been advocated and shown to pro-
vide cryoprotection with arguably lower toxicity. Both EG and PG
have been shown to provide cryoprotection at least as good as
DMSO in both human [61] and mouse ES cells [119]. In another
recent study using H9, a significant improvement in recovery of
hESCs was seen with PG when slow cooling was accompanied by
seeding [147].
Polyampholytes (polyelectrolytes bearing both cationic and
anionic repeat groups) such as poly-L-lysine have been used suc-
cessfully in the cryopreservation of rat mesenchymal stem cells
[148] and the vitrification of hiPSCs [149] and may offer yet
another alternative to DMSO. Recently, a commercial vitrification
solution, based on EG and containing poly-L-lysine, has become
available and has been demonstrated to provide effective cryo-
preservation [150]. As antifreeze proteins, they may also be a use-
ful additive in DMSO-based CPAs to help to control or reduce ice
crystallization.
Ectoine is a compatible solute first found in extreme halophilic
bacteria where it helps resist the effects of high salt concentration and
temperature by stabilizing the hydration shell of enzymes and mem-
brane proteins. It has recently been shown to provide improved, or at
least equivalent, protection to DMSO-serum-based CPAs when used
with methylcellulose and L-proline [151, 152] or in conjunction with
a low, non-cytotoxic, concentration of DMSO [153].
5.5 Cryo- Apoptosis in response to low temperature exposure is well docu-

preservation-Induced mented [154] and has been reported as a component in post-
Apoptosis cryopreservation cell death in a wide variety of cell types including
and the Role hematopoietic stem cells [155]. Heng and others [156–158] have
of Apoptotic Inhibitors demonstrated the involvement of apoptosis in the loss of cryopre-
served hES cells in the early post-thaw culture period with increased
production of reactive oxygen species (ROS) and the subsequent

activation of both the intrinsic and extrinsic apoptotic pathways.
The addition to the CPA of ROS-scavengers such as glutathi-
one has been shown to reduce cryopreservation-induced apoptosis
in murine ESCs [159]. Improved recovery in the presence of a
caspase inhibitor (caspase 1 inhibitor V) has also been reported
for MDCK cells [160] and for human embryonic stem cells,
stem cell-derived neurons and amniotic fluid-derived stem cells
(Z-VAD-FMK) [161–163].
5.6 Rho-Associated The propensity of human embryonic stem cells to undergo cell
Kinase (ROCK) death and differentiation when disassociated into single cells, even
Inhibitors without the added insult of freezing, has hampered the develop-
ment of efficient methods for scale-up and automation. New, milder
dissociation agents and improved passaging protocols have reduced
cell loss during culture but the introduction of Rho kinase inhibi-
tors has had a significant impact not only on the post-dissociation
culture of singlet cells but also on the post-thaw survival of hESCs
following cryopreservation. ROCK inhibitors have been shown to
significantly improve cell recovery, allowing single-cell suspensions
to be successfully cryopreserved using conventional slow cooling
protocols.
The Rho kinases are proteins that have been shown to play a
significant role in an array of cellular processes including adhesion,
proliferation, differentiation, and apoptosis depending on the cell
type [164] and the application of specific ROCK inhibitors to plu-
ripotent stem cells has been the subject of recent reviews [165,
166]. The ROCK inhibitors Y-27632 and Fasudil have been shown
to markedly reduce dissociation-induced apoptosis in hESCs dis-
sociated to single cells for passaging [167] and cryopreservation
[168]. Reports differ as to the benefit of including a ROCK inhibi-
tor in the CPA alone but its inclusion in the post-thaw culture
medium, or in both the CPA and post-thaw culture medium,
significantly increased survival with only a brief (24 h) post-thaw
exposure [169, 170]. Pinacidil, an FDA-approved vasodilator, is
also a ROCK inhibitor and has been shown to exert a similar ben-
eficial effect on hESCs during cryopreservation [171] as has the
protein kinase A inhibitor, H89 [172].
The exact mode of action in hESCs is a matter of some debate.
Recent evidence suggests a role in the suppression of cytokine
interactions and the MAPK pathway thereby reducing apoptosis
[173], while it has been postulated that instead of blocking apop-
totic pathways, ROCK inhibitors counteract anoikis (detachment-
induced apoptosis) or enhance cell-cell interactions through
modulation of gap junctions, thereby increasing the adhesive prop-
erties of the cells and enhancing post-dissociation (and post-thaw)
reaggregation [121, 174]. Whatever the mode of action, the
beneficial effect of ROCK inhibitors on reducing post-thaw injury
66 Charles J. Hunt
has been convincingly demonstrated for hESCs, hiPSCs, and MSCs

[175–177] although a negative effect of Y-27632 on post-thaw
survival has been reported for certain types of stem cell [178, 179]
and has been associated with aneuploidy and changes to cell
morphology [180].
5.7 Cryopreservation The maintenance of pluripotency is a major prerequisite for banking

and the Effect of cryopreserved stem cells. In a study using a “standard” slow
on Pluripotency cooling protocol with DMSO, Katkov et al. reported the loss of
the Oct-4 transcription factor (one of the canonical pluripotency
markers) following cryopreservation [181]. The study used a
genetically modified H9 cell line containing an Oct-4 promoter-
driven, enhanced green fluorescent protein (EGFP) reporter to
monitor Oct-4 expression as a surrogate marker for pluripotency.
The cryopreservation protocol was not optimized. Cells that sur-
vived the freeze-thaw process did not exhibit GFP fluorescence
within the first day of culture, with approximately 10% of the sur-
viving population regaining Oct-4 expression (as measured by
GFP fluorescence) by day 3 in culture.
Microarray studies using H9 have also been carried out on
cells post-thaw to identify processes and pathways that affect
post-thaw survival [182]. Cells frozen using a standard DMSO,
slow cooling protocol were subjected to global transcriptome
analysis at time points up to 48 h and showed a down regulation
of the pluripotency markers at all post-thaw time points from 3
to 48 h and a dysregulation of genes related to apoptosis, cell
death, and differentiation. However, Oct-4 levels in hESCs after
slow cooling in DMSO have been reported to be maintained
immediately post-thaw in the presence of the ROCK inhibitor
Y-27632 [170].
5.8 Low Temperature The potential for contamination by adventitious agents during
Storage both vitrification and storage of cells cryopreserved in open straws
and Transportation has already been discussed. Though less likely, contamination can
nevertheless occur with screw-capped cryovials as the caps do not
offer a leak-proof seal. Partial sealing, using commercially available
heat-shrink tubing, has been advocated; however, a completely
leak-proof seal is not guaranteed [183]. New, closed-system con-
tainers are becoming commercially available and have been
reported in the literature. The Cell Seal® system [184], a ported
cryovial sealed by conventional heat or RF sealers and Crystal
Zenith (CZ®) plastic pharmaceutical vials [185] have both been
assessed for cell cryopreservation and LN2 storage using hESCs.
CryoBioSystems, the manufacturer of the FDA-approved high
security straw, have recently launched a secure, heat-sealable cryo-
tube that is classified as a Class II medical device and applicable for
use in GMP-compliant cryopreservation systems.
A commercially available, gas-permeable cassette system for

scale-up has also been developed. These ported cassettes are com-
patible with DMSO (at least in the concentrations used in cryo-
preservation) and with storage in the gas-phase of LN2. Since they
are gas permeable, the cassettes can be used for the culture of hES
cells, and hESC colonies may be cryopreserved in situ by the sim-
ple expedient of changing the culture media for the CPA [186].
Master and working cell banks are likely to require long-term
storage below the glass-transition temperature required to arrest
molecular processes. This has generally been achieved by storage in
the liquid, or gas phase of LN2. Mechanical refrigeration, delivering
stable temperatures below −135 °C, is now reliable and studies on
stem cells indicate no significant differences between this and LN2 at
least in the medium term—up to 5 years [187]. In some systems,
temperature fluctuations around the glass transition have been
shown to be damaging [188, 189] and the storage of vitrified stem
cells material at these temperatures is not advisable.
Traditionally, storage at ultra-low temperatures was under-
taken by immersing the product in LN2 to ensure as stable a tem-
perature as possible. This practice has been questioned from a
cross-contamination perspective and storage in LN2 vapor- (or
more accurately gas-) phase is now considered best practice for
clinically relevant material. It has been recommended for gametes
and embryos [190].
The argument against storing in the gas phase above LN2,
particularly for vitrified material, is that storage vessels designed
originally for storage of product under LN2 show a considerable
temperature gradient when used to store product on a platform in
the gas phase. However, a number of technical solutions to this,
designed to be retro-fitted to older storage vessels, have been
developed which dramatically reduce the temperature gradient
[191, 192]. These technical “fixes” are commercially available.
Changes to the design of LN2 storage refrigerators, such as the
introduction of LN2-jacketed “isothermal” or “dry” vessels, as well
as vessels that are almost completely vacuum insulated have reduced
temperature differentials and storage temperature to below −180 °C
throughout the storage compartment.
Vitrified cells should be transported in LN2 dry-shippers and
not on pellets of solid CO2. Dry-shippers come in a range of sizes
from those suitable for use internally within a laboratory or cell
bank, to those for transporting cells over long distances. They
function by retaining LN2 within a molecular sieve-like material
that absorbs the LN2 and permits storage at LN2 temperatures for
up to 14 days. All require charging with LN2 before use. For con-
ventionally cryopreserved material, transportation in suitably
insulated boxes under solid CO2 is the accepted practice.
68 Charles J. Hunt
Studies investigating the potential for contamination have

shown that dry shippers do not pose a cross contamination
threat in the absence of damage to the shipment [193] and that
if contamination of the shipper occurs they are easily decontami-
nated [194].
6 Cryopreservation and Storage of Clinical Grade Stem Cell Lines
Over recent years more than 50 human embryonic stem cell lines
have been derived under conditions, and using media and media
components, which potentially make them suitable as starting
materials, for regenerative medicines [117, 195, 196]. Information
on hESC lines derived in the UK for clinical application can be
found in Ref. 197 and on the UK Stem Cell Bank website (www.
nibsc.org/ukstemcellbank).
The emphasis with all these clinical-grade cell lines has been on
the use of xeno-free reagents and the derivation and production of
the cell lines under conditions that comply with GMP.
Cryopreservation of these cell lines has utilized both slow cooling
[117, 196] and vitrification [195].
Most, if not all these lines, were derived and cultured on human
neonatal dermal fibroblasts from GMP-compliant sources and the
move to transition these cell lines to feeder-free conditions, more
suited to the expansion of seed stocks for clinical applications, is
ongoing. A number of protocols for the xeno-free culture of
pluripotent stem cells based on commercially available media have
been reported which also utilize commercially available cryoprotec-
tants [198, 199], including reports for some clinical-grade lines
[200]. Others have utilized DMSO deployed in the xeno-free
growth medium [201–203]. All of these protocols favor slow cool-
ing, though no attempt to systematically optimize the cryobiologi-
cal variables was reported in these studies.
All the reports, and others, on derivation of cell lines for clini-
cal application emphasis the necessity of complying with GMP
which forms the basis of regulatory frameworks for cells for human
application [204]. These regulatory frameworks are not specific to
pluripotent stem cells alone but relate to all cells and tissues for
human application regardless of the source material or the degree
of manipulation of the cells or tissue involved. To provide help and
guidance a number of Points to Consider documents, providing
guidance on areas of fundamental importance in developing seed
stocks of pluripotent stem cells for clinical application, are available
[205, 206]. These documents include sections relating to cryo-
preservation and low temperature storage.
7 Conclusion
Cryopreservation is a small but important process in the deriva-

tion, banking, and delivery of pluripotent stem cells for research
and clinical application. Without an optimal cryopreservation pro-
tocol and appropriate storage and transportation conditions, these
processes are compromised. Vitrification, while having been shown
to produce adequate recovery of pluripotent cells, has by no means
been optimized and questions regarding the suitability of the
method for scale-up beyond the laboratory setting, and its accept-
ability for clinical application from a regulatory perspective, remain
largely unanswered.
The improvement in slow cooling protocols, the use of con-
trolled nucleation and apoptotic inhibitors coupled with new
GMP-compliant container systems and cryoprotectants has the
potential to provide a cryopreservation process compatible with
regulatory requirements, scale-up, and automation once these
cryopreservation protocols have been optimized.
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52:49–55 clinical applications: International Stem Cell
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human pluripotent stem cells in defined medium. Suppl):1–44
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1516:47–56 Ltd, pp 167–185
Chapter 6
Quality Assured Characterization of Stem Cells

for Safety in Banking for Clinical Application
Kevin W. Bruce, John D.M. Campbell, and Paul De Sousa
Abstract
The promise of human pluripotent stem cells to serve as a scalable and renewable starting material for “off
the shelf” therapeutic cell products to repair or replace cells and tissues damaged by disease or injury is
unparalleled. Whether originating from embryos or the genetic manipulation of adult tissue-derived cells,
this prospective impact dictates a comprehensive yet practicable standard of quality assured characteriza-
tion, blending existing and bespoke standards and considerations. Here, we provide a guide to qualifying
the suitability of this resource for human clinical application.
Key words Pluripotent stem cell, Stem cell banking, Safety, Cell identity, Characterization, Quality
control, Procurement, Quality assurance
1 Introduction
In the last decade and a half, the ability to isolate human embry-
onic stem cells (hESCs) and induce pluripotent stem cells (hiPSCs)
from somatic cells has driven a surge of interest in the establish-
ment of banks of such cells and the associated primary cells from
which the latter can be generated for research, industrial, and clini-
cal application. Not surprisingly for an emergent field the tech-
niques and processes used to generate these cells have varied greatly
depending on the starting material along with the resources and
expertise available locally. For clinical use, there is also variation in
regulatory process and standards, and differing ethical consider-
ations influencing their implementation. In the face of such diver-
sity there is a clear necessity to attempt to define and adopt best
practice and standardized methodologies for the characterization
of such banks to provide reliable and comparable benchmarks from
which to conduct research and develop therapies [1, 2].
There are a few examples of deriving stem cell lines with the
intention that these may ultimately be used as the starting material
for differentiated cellular therapeutics [3–6]. The standardization
79
80 Kevin W. Bruce et al.
of processes and the identification, characterization, and functional

testing of such stem cell lines is critical since any final therapeutic
product will be intrinsically linked to its starting material. A thor-
ough understanding of how the cells should look and behave is
essential to be able to demonstrate control over any subsequent
process the cells are used in and any changes made thereafter.
It is essential that any cell bank providing quality assured cells
can demonstrate that the cultures have specific characteristics and
that they are free from microbial contamination. It is therefore
necessary to implement an appropriate testing regime that can
establish key criteria for quality control of cell lines such that their
characteristics can be demonstrated.
Quality assured (QA) characterization of stem cell banks is also
critical to enable robust stem cell research, the outcome of which
will ultimately inform the development of safe and efficacious stem
cell-based therapies. Robust banking and distribution of cell lines
requires defined processes and testing regimes to provide consis-
tency and confidence as well as permitting comparable develop-
ment across laboratories. Characterization to ensure quality, safety,
and efficacy must include testing at critical stages identified
throughout the banking process including early outgrowth stages,
during culture expansion, pre- and post-cryopreservation and
include stability and viability assessment after banking.
Extensive standardization of stem cells as a primary resource
provides a baseline from which to conduct efficacious research and
development with confidence. The approach that is taken to test-
ing cells must consider the differing stages of the banking process.
Testing should permit an analysis of how the cells behave both
during their initial expansion and following their recovery from
cryopreservation. In this regard, it is important to keep the bank-
ing process as consistent as possible, but also to be in control of any
changes, be aware of their impact and to consider and evaluate the
effect these changes may have on the cells.
Banking processes will vary, particularly in relation to the type
and origin of the starting material. There are obvious differences to
consider between the procurement of embryos for the derivation
of hESCs and the procurement of adult tissue to establish banks of
somatic cells.
It is crucial that any laboratory wishing to conduct thorough
QA characterization testing does so within the framework of a com-
prehensive Quality Management System (QMS). This will facilitate
control over the establishment and implementation of any test
methods and data generated, and will allow for managed changes to
be implemented successfully. An established QMS should minimally
cover raw materials and other processing components, consum-
ables, equipment (including use, maintenance, and repair), meth-
ods (processing and testing) documented as Standard Operating
Procedures (SOPs), data recording, and methods for reporting.
Quality Assured Characterization of Stem Cells for Safety in Banking… 81
Where stem cell lines are being banked with the intention to
use them in the development of cell therapies, it is essential that
this is performed with an understanding of the relevant legal and
regulatory requirements. Under current European regulation, plu-
ripotent stem cell-derived cellular therapeutics are most likely to be
regulated as advanced therapeutic medicinal products (ATMP).
Current best practice is to qualify raw materials, and to produce
and characterize banked cell lines to meet these exacting standards
from initiation of the cultures. This will ensure that this part of the
regulatory process starts off on a sound footing regardless of the
final use of the banked cells.
The characterization data generated for a stem cell line forms a
critical component of the complete history of a cell line and should
be compiled appropriately to provide documented evidence for
this purpose.
This chapter will review some of the key aspects related to the
characterization of stem cell banks with a focus on demonstration
of their safety and efficacy in the context of their intended use in
the manufacture of cell-based therapies. This is structured in terms
of describing the overall approach and strategy followed by a
description of cellular characterization (identity, purity, potency,
viability, and stability) and microbiological testing.
2 Procurement
The ability to clearly demonstrate the provenance of the original

material is a critical component that underpins all downstream ele-
ments. The quality of any cells used as starting material ultimately
for use in clinical application can only be determined where there is
a full and traceable understanding of its origin. The potential risk of
transmission of infectious agents or genetic abnormalities, the need
for comprehensive and fully informed consent, and robust traceabil-
ity are the primary issues to control. Proper planning, specifications,
recording, and documenting of procurement activities are critical.
Where donated cells and tissue are used it is essential that pro-
curement is conducted only following ethical review of procedures
and with fully informed consent from the original donor(s) [7–
12]. It is critical to ensure that the donor specifically is aware of the
intended research and clinical uses of the cells derived from their
donation and that these cells are likely to be shared with others
globally, may be transplanted into humans and animals, and may
be exploited commercially. The extent of testing to be performed
on the cells/tissue donated and any cells derived thereafter such as
human embryonic or induced pluripotent stem cells should be
made clear. This will include testing for pathogens, genetic testing
including whole genome sequencing and, although challenging,
should also make donors aware that future testing methods that
may not be currently available, but are feasible in the future. All
donations should be anonymized to maintain donor confidential-
ity; however, they should be coded and traceable. Anonymized
copies of the original consent documentation should be obtained
as a record of the consent.
Where any specific component of the procurement process is
being undertaken by a third party, then suitable specifications and
agreements should be in place to describe and control the activity.
Donor selection criteria may be strategically based on specific
factors such as ethnic background or immune haplotype [13].
However, from a quality perspective there are a number of other
inclusion/exclusion factors that must be considered.
The risk of microbiological contamination (bacterial, fungal,
viral, parasitic) should focus on those agents most likely to be con-
taminants in relation to the geography, donor cohort, and cell or
tissue type being procured. Ensuring that donations meet all sig-
nificant acceptability criteria for local/international blood or plate-
let donation [14] is one strategy for managing donor eligibility/
exclusion criteria. Any approach should include mandatory testing
for the most commonly known and transmitted pathogens and
indicators of an immunological response such as Syphilis, HBV,
HCV, HIV (genome), as reflected by the detection of serological
antibodies to these pathogens, as well as any other pathogens clas-
sified as at higher risk of prevalence in the country of origin. It is
important to ensure that testing for these markers is performed on
samples taken at the time of donation and performed using tests
having undergone validation for use with donor samples providing
suitable specificity, sensitivity and with suitable controls.
Donors should not be considered suitable for donation if, for
example they:
1. Were unwell at the time of donation. Deferral periods for spe-
cific infections may exist and/or vary in accordance with local
regulatory guidelines for other cell and tissue products (e.g.,
for blood donors).
2. Have tested confirmed repeat positive for infectious disease
(see the list above and section 5 below).
3. Have received a blood transfusion within the previous
12 months (it is advised to check local regulatory guidance for
variations and risk factors).
4. Regarding TSE/CJD risk: has had treatment with pituitary
extracts of human origin; has received corneal or dura matter
transplant, has a personal or family history of CJD.
5. Donors with serious active, chronic, or relapsing disease (e.g.,
cardiovascular disease, gastrointestinal, genitourinary, hema-
tological, immunological, metabolic, renal, or respiratory
diseases).
6. Have been or are intravenous/intramuscular drug users.

7. Has received a xenotransplant.
8. Persons whose sexual behavior puts them at high risk of
acquiring severe infectious diseases.
It is important to keep a continued watch over new and emerging
infections and consider their potential to be transmitted in the
context of the cell type(s), process, and quality control testing
implemented. The greatest concern will be for diseases for which
there are no validated tests available to identify pathogen presence
in the absence of visible symptoms of disease, or technology to
remove infectivity if detected. Of these, prion diseases of human
origin (Creutzfeld Jacob Disease, Kuru) or animal origin (e.g.,
Bovine Spongiform Encephalopathy, Ovine Scrapie, Chronic
Wasting Disease) evident globally, constitute one of the most sig-
nificant concerns for which risk assessment-based geographic
deferrals have been implemented to date for established human cell
and tissue products. Assessments should consider the general
donor background as well as the cell type being procured, for
example using cells originating from the CNS may pose a higher
risk. Potential donors who are clinically ill and those with familial
instance of prion disease are assumed to be excluded from eligibil-
ity to donate. Similarly, individuals at risk following medical treat-
ments (e.g., surgery) should be identifiable and will have been
informed making them identifiable. The largest risk is likely to
come from transmission of variant Creutzfeldt-Jakob disease
(vCJD) arising from infection from bovine spongiform encepha-
lopathy (BSE) and other cattle sources. Only blood and blood
products have so far been implicated in actual cases of transmis-
sion. This transmission risk is currently managed by the use of
leuko-depleted blood products, e.g., in the UK. Maternal trans-
mission of prion disease has never been established despite cases of
pregnancy in affected mothers [15]. Interestingly, limited investi-
gation to date has suggested that if hESC are exposed to an infec-
tious prion inoculum, protease resistant prions are taken up but
then rapidly extruded by cells and not perpetuated during further
cell growth [16].
When acquiring samples to generate stem cells for clinical
application there should be mechanisms by which ongoing donor
traceability can be made. Traceability should be considered in
terms of tracking back to the original donor and their medical
records in a suitably confidential and consented manner, and in
relating to forward traceability during banking activity and any
subsequent clinical manufacturing and distribution processes. Such
mechanisms must be appropriate in scale and suitably coded so as
to maintain donor confidentiality. The ability to trace forward and
backward from donor records is important mainly from a quality
perspective, for example should the donor subsequently be
diagnosed with a disease (e.g., CJD or hepatitis C), or in case re-

consenting is required to enable unforeseen activities. Guidelines
and standards now exist for implementation of suitable coding of
cells and tissue intended for clinical application [17, 18] and should
be referred to.
For induced pluripotent stem cells there are a number of spe-
cial considerations.
It is important that the characteristics of the starting material
are documented and also that archived material is available should
the need arise to perform comparative or confirmatory analysis.
To this end, archive samples at suitable volumes for both cellular
and genomic material from early tissue or cell populations should
be taken and stored appropriately.
Cell storage, packaging, and transport criteria should be clearly
defined for the samples that are obtained. Temperature limits, tim-
ing restrictions, storage locations and containers, and appropriate
alarms should be specified. Biosample containers as well as ship-
ping vessels should be inspected and records of transport must be
maintained.
3 Testing Within Defined Cell Banking Procedures
Banking processes will vary and it is essential that each process for
different cell types is fully understood. The use of flow diagrams
for processes can provide useful tools to identify critical stages and
permit the targeting of testing time-points. Any cell bank should
ensure that it employs a documented Master and Working Cell
Bank system (Fig. 1).
The procurement process, including the critical stage of pro-
viding information and obtaining consent in an ethical and robust
manner, should always be considered the beginning of the banking
process (see Chapters 3 and 8).
A typical banking process involves the expansion of cells to the
Pre-Master Bank (PMB) (or Seed Lot) stage, followed by further
expansion to the Master Cell Bank (MCB) stage (Fig. 1). Aliquots
from the MCB are then expanded to the final stage of a Working
(or Distribution) Cell Bank (WCB). Further WCBs are then man-
ufactured by returning to and expanding further aliquots from the
MCB. The PMB acts as an original stock from which subsequent
MCBs can be derived, although it should be considered a precious
resource that is rarely required. It is important to design the
banking strategy and scale of each bank stage according to the
anticipated scale of production of final clinical cell type to be imple-
mented, taking into consideration, for example, the expansion and
attrition rate in downstream processing and the anticipated clinical
dose and number of doses required.
Fig. 1 Schematic of a banking workflow. Cell scale-up is performed to generate a Pre-Master Bank (PMB)
(or Seed Lot) stage, followed by further expansion to the Master Cell Bank (MCB) stage. Aliquots from the MCB
are then expanded to the final stage of a Working (or Distribution) Cell Bank (WCB). Additional WCBs are manu-
factured from aliquots of the MCB
In cases where early stage stem cell populations are being

derived and subsequently supplied to a cell banking facility, it is
essential that there are procedures in place for the receipt and quar-
antine of such materials until such time as testing can be performed
to ensure there is no risk of contamination or infection.
The banking process avails a number of stresses on the cells
being expanded, for example age (cell doublings), passaging, and
freeze/thaw cycles. This requires that a defined Quality Control
(QC) regime is implemented that monitors the cells for stable con-
tinuity of their desired characteristics and to test for the absence of
adventitious agents.
Critical control points should be identified in the banking pro-
cess and suitable tests identified at each point to both characterize
the cells and test for the absence of adventitious agents. At each
such Sample Point it is very important to consider the quantities of
material required for testing, especially where live cells are required,
to be sure sufficient cells are available to conduct testing homoge-
nously at a single passage number, or cell doubling stage. A variety
of sample types will be needed to conduct all suitable tests. For
example, samples of spent media will be required to test for myco-

plasma and endotoxin, whereas live cells will be required from
both before and after cryopreservation for a number of character-
ization tests. In addition, samples of cryopreserved cells may need
to be used to test for sterility, or to extract nucleic acids for testing,
and also to be held as archive samples for future testing.
In general, more extensive testing is performed near the begin-
ning of the process and, where justified, less testing can be carried
out at the latter stages. An example of this kind of approach would
be to screen for the presence of any viral contaminants as early in
the process as possible. Further viral testing downstream would
only be required if there was potential risk of introduction of con-
taminants from components used in the manufacturing process,
for example when products from sources of animal origin are used.
This kind of pyramid approach also helps to prevent investment in
expanded culture of cells that are contaminated or display unde-
sired characteristics.
Fully traceable documented evidence of the banking proce-
dure, compiled for example as a Cell Line History File, is essential
to be able to demonstrate that procedures have been followed and
to allow for troubleshooting should any issues arise. Such docu-
mentation should be implemented early and record all activities
including procurement data and the starting cell material, raw
materials and reagents, facility and equipment and qualification
status, procedures and protocols followed, testing performed and
storage and distribution information. These documents should be
updated regularly.
4 Cell Characterization
Cell characterization will primarily be considered an evaluation of

cell characteristics indicative of level of safety. In the main, such con-
siderations revolve around microbiological risks, but will also include
a number of other factors concerning function and stability. This
section and Table 1 describe the methodologies that can be imple-
mented to evaluate cells within a structure cell banking system.
4.1 Genetic Identity Establishing and monitoring the identity of cell populations is criti-
cal for quality and safety [19], particularly where a specific popula-
tion of cells is claimed to have been isolated form a heterogeneous
starting population. Obtaining a genetic profile of the cells provides
evidence that a cell line is monoseptic and can be compared to data
from donor material or primary cells to confirm its origin.
Typical forensic methods are carried out using PCR amplifica-
tion of short tandem repeats (STRs) and commercial kits are avail-
able for this purpose [1]. Each STR represents a short region in the
genome that can exhibit variations in terms of length between indi-
viduals. These STRs are found across the entire genome. The STR
Table 1
Details of appropriate testing technologies
Classification Example test method Purpose

Identity Microsatellite PCR (mPCR) DNA profiling to determine the genetic
signature and sex/species of the cell line
Phenotype Immunocytochemisty To assess levels of staining for a panel of
pluripotency markers (e.g., Oct 4, Nanog,
Sox2, Tra-1-60, Tra-1-81, SSEA3, SSEA4,
SSEA1 [negative])
Flow Cytometry Assess antigen levels and cell surface markers
commonly associated with hESC (TRA-1-81,
SSEA-4) and transcription factors (Oct-3/4,
Sox2)
Viability Quantification of cells by, e.g., Assess viability of culture prior to
Alkaline phosphatise, trypan cryopreservation
blue exclusion, flow cytometry
Short Tandem Repeat (STR)
analysis
Genotype Karyology (G-Banding), Confirmation of normal ploidy by G-banding
Spectral Karyotype (SKY) Identification structural chromosome
Single Nucleotide Polymorphisms aberrations
(SNP) Copy number analysis of specific DNA regions
HLA Tissue Typinga To establish the HLA Class I and II type
of the line
Pluripotency In vivo teratoma formation To establish the capability of the line
to form the three germ layers in vivo
In vitro embryoid bodies To establish the capability of the line
to form the three germ layers in vitro
Morphology Photography To capture a visual record of the line
Microbiology Mycoplasma Ph. Eur./ Ph. US Mycoplasma testing to EP/USP guidelines
and virology in vivo and in vitro viral screening Testing for absence of HIV, HBV, HCV, EBV,
tests hCMV, HTLV 1, Syphilis
NOTE: Additional viral tests should be included
as identified from risk assessments in relation
to, for example, geographic background of
donors, process components.
Sterility Sterility testing to EP/USP guidelines
Ph. Eur./ Ph. US
a
Can also be used as part of identity testing
loci and the number of STRs analyzed varies internationally, but
some are common. There are a variety of methods used for forensic
analysis, but analysis of between 11 and 15 STR loci is considered
standard practice. This method is one commonly used internation-
ally and so enables rapid confirmation by other labs that they are
using the correct cell line. It is relatively cheap and high-through-
put, but requires specific equipment and trained staff to perform the
work and analyze the data. Standards are available to ensure a robust
approach [20].
Other methods, such as the use of Single Nucleotide

Polymorphisms (SNPs) or Humam Leukocyte Antigens (HLAs),
can be used, but may not provide data that is easily comparable
across laboratories [21].
When conducting such genetic testing on human cell lines it is
always important to consider how the data is recorded and distrib-
uted to ensure appropriate donor confidentiality is maintained.
The publication of data that may allow the identification of specific
donor individuals could have implications for them and their fami-
lies’ future healthcare [22].
4.2 Phenotype Testing the phenotype of the cell population, by screening for the
presence or absence of cell surface or intracellular markers, pro-
vides essential characterization data. Phenotype can be established
in a number of ways. The most common method is the quantifica-
tion of multiple known cell surface markers of pluripotency by flow
cytometry. Flow cytometry is a high-throughput system that uti-
lizes the principles of light scattering, light excitation, and detec-
tion of emission of light from fluorescent dyes to provide
multiparametric measurements of a single flow of particles or cells.
Information is simultaneously collected on cell size, granularity,
and protein expression using antibodies conjugated to fluorescent
dyes. Additionally, information can be collected on cellular viability
using vital dyes and cell cycle using DNA-binding fluorescent
probes. Modern Flow cytometry uses multiple fluorochromes and
can therefore allow a number of cell characteristics to be detected
simultaneously. A simple set of parameters can be determined to
measure cell cycle, viability, and phenotype in a 4–6 color single
panel for routine monitoring at various points in the production
process (see, for example, Fig. 2). More extensive characterization
with a comprehensive marker set can be carried out in the cell
banking process. A typical marker profile for pluripotent cells
would include positive expression of OCT-4 (POU5F1), Sox-2,
Tra-1-60, Tra-1-81, SSEA-3, and SSEA-4, with low or negative
expression of SSEA-1 [23, 24]. Immunocytochemistry can also be
used to provide additional information on the morphology local-
ization of specific antigens.
Recording the visual morphology of stem cell cultures can pro-
vide valuable data to support phenotype determination, particu-
larly where adherent cells are concerned. It can also provide useful
information on growth profile and anticipated confluency and pro-
cessing time points. This type of data should always be used as an
additional methodology and not as a stand-alone test.
Phenotype can also be determined using molecular markers of
gene expression associated with a specific stem cell type. For human
pluripotent stem cells, the expression of known pluripotency genes
such as Nanog, Oct-4, DMNT, TDGF, and GABRB3 can provide
very useful information [23]. However, such RNA expression data
should always be confirmed at the protein level. The use of Protein
1e3 1e3
a b
1e2 DRAQ7 1e2

APC-Cy7-A
FSC-H
1e1 1e1
1 1
0 0
-1 -1
-1 0 1 1e1 1e2 1e3 -1 0 1 1e1 1e2 1e3
FSC-A FSC-A
1e3 1e3
c >98% d
<1%
1e2 1e2
APC SSEA4-A
APC SSEA4-A
1e1 1e1
1 1
0 0
-1 -1
-1 0 1 1e1 1e2 1e3 -1 0 1 1e1 1e2 1e3
FITC TRA160-A PE SSEA1-A
Fig. 2 Flow Cytometric Analysis of the RC9 hESC line [25]. (a) —live cells are selected for analysis by selecting
cells with High Forward Scatter (FSC), and cells that exclude the Vital Dye DRAQ7 (measured in the APC-Cy7
channel). (b)—The live gate is activated and doublets are excluded from the analysis by plotting FSC—Area
versus FSC-Height, and excluding events that fall below the diagonal. (c)—Live singlet cells highly express the
characteristic SSEA-4+ TRA-1-60+ phenotype >98%++ (d)—The phenotype of the cells is confirmed by
analysis of SSEA-4 versus SSEA-1, and less than 1% of SSEA4+ cells express SSEA-1. MACSQuant 10 flow
cytometer, MACSQuantify software 2.6. Gate line thickness highlighted for presentational purposes, percent-
ages generated using original gates in the analysis software
Ligation Assays (PLAs) can provide a measure of protein expres-

sion which can be directly correlated to the mRNA and/or miRNA
expression. In these highly sensitive PLA assays a paired set of
antibody-oligonucleotide probes bind to the protein target. A
third oligonucleotide then hybridizes to the oligonucleotides pair
when they are bound in close proximity. This ligation product can
then be specifically amplified and detected by PCR to provide a
measure of the level of expression of the specific protein. This
method can be expensive and require significant optimization.
It is important to remember that it is the overall expression

profile of a given set of markers that is important since other cell
types may express one or more of the known pluripotency
markers.
More recently, the use of micro RNA profiles has been used
where such profiles can be linked to specific cells/tissues.
Confirmation that the cell population exhibits all known phe-
notypic markers for pluripotency should not allow one to allude
that the cells will be pluripotent. Additional potency measurements
must be made to confirm this.
4.3 Cell Function or The biological potency of the cell population should be demon-
Potency strated. For pluripotent cells the ability to produce cells representa-
tive of all three germ layers should be tested for. This has
traditionally been performed by testing for the ability of the cells to
form teratomas in SCID mice. This technique can be variable,
although attempts have been made to standardize the procedure
[26], and different results have been seen depending on criteria
such as: number of cells injected, site of injection, media/matrix
components used as injection vehicle and over numbers of repli-
cates. In this setting, it is important to consider the relevance of
any negative results. While a cell line that forms teratomas repre-
sentative of all germ layers may be chosen for its flexibility, a cell
line that consistently differentiates down a single pathway may
prove to have considerable utility for making cells of a specific type.
In this case, arguments could be assembled to support increased
safety due to reduced risk of spontaneous differentiation in unde-
sired ways.
Alternative in vitro methodologies for assessment of pluripo-
tency do exist. For example, formation of embryoid bodies where
aggregates of pluripotent cells are cultured so that cells representa-
tive of the three germ layers spontaneously arise. These can then be
assessed by immunocytochemistry or RT-PCR for detection of
markers of differentiated cell types. More recently, protocols for
the directed differentiation of pluripotent cells have become avail-
able; however, these have been criticized as being less predictive of
full pluripotency as they may tend to give rise to differentiated cell
types of a more specific pathway as compared to the spontaneous
differentiation seen using teratomas or embryoid body techniques.
Whilst such directed differentiation methods may provide a better
indication of terminal cell or tissue differentiation potency, they
vary greatly in methodology, can take time, be costly and difficult
to standardise.
Additionally, some tests are available which consider the
transcriptome-based profiles of the cells themselves [27, 28] and
compare these to a database of other cell lines “validated” in terms
of other supportive data. Such approaches can be useful, but in the
absence of actual demonstration of in vitro or in vivo differentia-
tion of the cells will always be open to question.
In summary, there is no single method that will provide a suit-

able measure of potency or function and one or more methods will
need to be selected based on the specific requirements in each case.
For pluripotent cells it is important not only to show ability to dif-
ferentiate into all three germ layers, but also that the cells can com-
mit to specific mature cell types demonstrating anticipated mature
phenotypes and functions.
4.4 Purity During expansion it is critical that the cell population is monitored
for purity. Purity data should be evaluated in the context of the
data generated for identity. Purity analysis should check for evi-
dence of unwanted cells, for example differentiating cells in a plu-
ripotent cell culture, as well as for the expected cell type. Flow
cytometry can be used here to examine the detail of cell line
heterogeneity, and flow cytometry demonstration of the lack of
contaminating cells is often of high importance. Where labs are
processing multiple cell lines it is prudent to check for evidence of
any cross contamination. Limits should be set for acceptable num-
bers of each undesired cell type, taking into account the level of
risk the cell type presents in the context of the intended use of the
cell line being processed.
Specific cell types may also require additional tests, for example
induced pluripotent stem cell populations should be tested for the
absence of any residual vector components used for reprogram-
ming, for example by PCR for detection of exogenous reprogram-
ming factors.
Where a feeder cell population has been used in coculture it is
necessary to check that their removal has been successful when the
desired population is harvested. In some cases, removal of feeder
cells (e.g., by the use of antibody-conjugate magnetic beads) may
be desirable or useful.
Purity should also be considered in terms of culture compo-
nents and excipients used as well as the cells. Purity tests should
also consider any potentially harmful components that might rea-
sonably be expected to have been introduced by the manufactur-
ing process. For example, antibiotics, matrix components,
breakdown products from raw materials.
4.5 Viability/Growth The viability of the cell population should be checked at regular
Profiling intervals and after changes to cell culture conditions. In particular,
viability should be assessed following recovery from cryopreserva-
tion, being mindful that cells should be assayed following some
time in culture after recovery to avoid false overestimation of via-
bility. Limits should be placed on acceptable percentage of nonvi-
able cells at recovery from cryopreservation and passaging.
As well as simple cell counts alternative methods such as alka-
line phosphatise detection [29] or trypan blue exclusion can be
used; however, these methods can present inherent variability
between operators in terms of performance and experience of

assessing results. This can be overcome by introducing automation,
but this adds cost and requires larger sample volumes. Methods for
assessing cell viability by flow cytometry are extremely accurate and
simple to perform—DNA-binding vital dyes such as propridium
iodide or DRAQ7 allow for accurate determination of viability. This
method is powerful as the cells can be gated to identify debris and
cell aggregates in the culture. A variety of dyes that bind to DNA
and are not affected by cellular fixation have also now been devel-
oped, and these allow accurate viability determination even when
local procedure requires cells to be fixed before analysis.
Monitoring the growth profile of the population is important
to provide a relatively easy indicator of transformational change in
the population. This measurement should be taken regularly and,
for example, after adaptation to new culture environments or pas-
saging regimes. Methodologies for measuring growth profiles will
vary depending on the nature of the cultures. For example, sam-
ples of cells passaged as single cell suspensions can be measured
using cell counters. Cell populations growing in colonies or clus-
ters are more problematic. Some automated imaging systems are
now available which can be useful for these cells [30].
4.6 Genetic Stability Cell lines will have some inherent variability in culture, particularly
and Karyotype at high passages [31]. However, it is important to maximize stabil-
ity by minimizing the number of population doublings and pas-
sages, being consistent with culture media, passaging regime and
process methodology, and controlling and monitoring the culture
environment. Although stability should be evaluated using data
from all cell testing including proliferation characteristics, genetic
profile, phenotype, and response to stresses such as cryopreserva-
tion, it is important to regularly karyotype cells by, for example,
G-banding. Testing should be performed early to establish a base-
line of “normality” for that line. Changes to the genetic makeup of
a cell line can lead to loss of function or uncontrolled differentia-
tion. Increased regularity of testing should be performed where
there are changes in culture conditions, following recovery from
cryopreservation and at high passage numbers and passages beyond
that of the MCB and WCB.
Analysis of the karyology of cell lines by G-Banding is the most
common method routinely applied in cell banking. It is a well-
practiced and cheap method used to assess the chromosomal status
of a cell line in terms of the number and appearance of the chro-
mosomes. This method requires that mitotic cells are arrested in
the metaphase stage of division when the chromosomes are at their
most condensed. The arrested cells are then treated to release the
chromosomes from the nucleus and are fixed to a glass slide. The
fixed chromosomes are then treated with an enzyme (e.g., trypsin)
to degrade chromosomal proteins and “relax” the chromosomes
Fig. 3 Karyogram image of iPSC line RCi002-A. G-banding image of iPSC line RCi002-A demonstrating normal
46, XX chromosome complement, and banding pattern from 20 of 20 spreads
that can then be more easily stained and evaluated under a micro-
scope. Usually, Giemsa (lending the “G” to G-banding) stain is
then applied which binds to A T-rich areas. Once visible the stained
bands make it easier to identify chromosomes that are arranged as
an ideogram for presentation. The final stage of evaluation of the
chromosomes requires the skills of an experienced cytogeneticist.
More recently, individual elements of the preparation processes
have been automated leading to increased standardization of chro-
mosome quality, important in assays such as these where operator
to operator variability can be high. See example in Fig. 3. Flow-
cytometry-based DNA analysis can also detect abnormalities in the
DNA content of cells, is easily standardized, and has the advantage
of rapidly examining thousands of cells, but also requires experi-
enced operators for accurate results.
Similar to G-banding, but with increased resolution, is spectral
karyotyping (SKY). SKY utilizes fluorescent probes for DNA
sequences on specific chromosomes to label the sample. The fluo-
rochromes are then visualized for analysis.
It is important to be aware that the sample size and the sensi-

tivity of the method chosen will impact the sensitivity of detecting
abnormal clones. With hESCs for example, at least 20 metaphase
spreads should be prepared for routine in-process testing. For
assessment of banked material intended for clinical application this
number should be much higher (ca. 50-100 spreads). All spreads
analyzed for human cells should show diploid chromosomes with
no structural abnormalities detected. When an abnormality is
detected it may be necessary to repeat testing of the same culture,
or at subsequent passages to determine if the result is spurious, or
representative of the population.
There are many new technologies for genetic analysis which
provide rapid analysis and provide unprecedented levels of sensitiv-
ity and resolution. The cost and speed of performing these tests are
rapidly improving. Techniques such as SKY, Comparative Genome
Hybridization (CGH) [32], and analysis of Single Nucleotide
Polymorphisms (SNPs) can provide high resolution. Additionally,
whole genome sequencing is becoming more cost efficient. These
methods can produce significant quantities of data and it is impor-
tant to ensure that sufficient resources and expertise are available
to manage the results correctly. Also, the extensive data generated
will be challenging to handle and raises concerns for the mainte-
nance of donor confidentially.
The analysis of single nucleotide polymorphisms (SNPs) is an
alternative method that increases resolution compared to
G-banding and performs copy number analysis of specific genomic
regions. This method has been suggested for use in clinical applica-
tions [33, 34]; however, the significance of any gain or loss is chal-
lenging to determine. This approach would be useful where
predetermined SNPs in regions of known risk, e.g., tumor sup-
pressing genes, were targeted.
It is important to remember that with all these techniques the
number of cells analyzed will directly affect the sensitivity of the
assay performed and how representative of the results generated
are of the population sampled.
5 Microbiological Tests
The risks of microbiological contamination from a number of

sources need to be considered. It is important to consider the risk
from donor individuals providing primary tissue for cell derivation,
the risk from manufacturing components used, and the risk from
the manufacturing environment. In most cases, it will be possible
to test raw materials, the cell populations, supernatants, or spent
media for the presence of adventitious agents; however, in some
cases, a risk-based approach may be required which leans on data
from parametric testing and information.
The presence of microbial contamination presents obvious

risks in the context of manufacture of clinical products. It is also
necessary to be aware of the impact such contamination can have
on the biological characteristics of the cell population being cul-
tured. For example, a low level viral infection may not have a sig-
nificant impact on cell death, but could dramatically influence
biological activity. This type of contamination can impact not only
the suitability of the cells for clinical use, but also any research data
being generated.
Taking a holistic view of processes from donation and procure-
ment, through preparation of culture reagents and equipment, to
maintenance and cryopreservation of cultures it is important to
establish microbiological testing at critical points in the process. In
addition, it is advisable to introduce procedures to minimize risk to
other established cultures. For example, it is good practice to main-
tain quarantine procedures for primary tissues or cell lines newly
brought into the laboratory. Such cultures should ideally be main-
tained in a dedicated laboratory, but minimally in segregated
equipment until sufficient data is available to justify their
relocation.
Test methods used for microbiological testing should always
be qualified and it is important to be sure that appropriate levels of
sensitivity, specificity, and robustness are being used in respect of
testing cell cultures. It is equally important to be sure that sam-
pling regimes ensure results are representative of the cell culture,
or cryopreserved cell bank as a whole.
5.1 Primary Cells As with any application of human-derived products for use in ther-
and Tissue apy, the risk of transmission of viral contamination from the origi-
nal donor is the most obvious. It is critical that planning is done
well in advance of procuring any primary tissue to determine rele-
vant inclusion/exclusion criteria for donors that may influence risk
of infection as well as the types of medical and lifestyle information
that will be useful for selection of donors and risk mitigation.
Medical screening of donors should be performed at the point
of donation. Where medical screening information is available it is
still necessary to take caution with any donated tissue as there it
will likely only be possible to test for the most common infections
and a certain level of unknown infection may remain, or have been
obtained between screening and donation. The use of lifestyle
questionnaires, such as those used in blood donation centers, may
be useful to help reduce risk. Such questionnaires must be used in
the context of what is ethically and morally acceptable to ask. This
is especially true where tissue is being donated in special circum-
stances, for example where embryos are being donated from cou-
ples undergoing assisted conception procedures it might not be
appropriate to ask questions around individual sexual history.
5.2 Viruses Early and comprehensive testing of material is critical to preventing

investment in contaminated product. This is especially true for
viruses as subsequent testing can justifiably be excluded based on
process controls and use of suitably sourced and tested raw
materials.
It will not be possible to test for all viruses and a risk-based
approach should be taken to test those of highest risk given the
origin and history of the cells to be tested. Typically, viruses of
blood-borne origin should be prioritized. In addition, it is neces-
sary to consider the risk from raw materials used, for example the
use of fetal calf serum, animal sourced growth factors, or enzymes
can pose a risk of transmission.
If possible, the expertise of clinical and microbiological special-
ists should be drawn on to enable a risk-based approach to be taken
with regard to selection of the most relevant viruses to test for. In
general, when testing established cell lines some of the most com-
mon viruses to test for include HIV I/II, HepB, HepC, hCMV,
HHV 6-8, CMV, HTLV I/II.
It is critical that procedures are established which describe
actions to be taken in case of a positive result for any virus. These
procedures should also be complimented by routine practices to be
taken by operational staff to minimize the risk of transmission.
5.3 Mycoplasma Tests for the presence of bacteria, yeast fungi, and mycoplasma
and Sterility should be conducted routinely. The use of antibiotics should be
eliminated as soon as possible; however, where they are used in the
culture medium, these should be removed prior to sampling.
Mycoplasma is recognized as a common contaminant of cell
cultures due to the risk of contamination from numerous sources.
Three major sources of contamination are (1) other cell cultures
introduced to the laboratory; (2) humans (M.orale, M.fermentans,
M.salivarium, or M.hominis); and (3) cell culture reagents such as
bovine sera (M.arginini, A.laidlawii) or porcine trypsin
(M.Hyorhinis). Mycoplasma can be very difficult to remove from
cell culture as their small size limits filtration and they can be dif-
ficult to detect without establishing routine testing procedures.
A pharmacopoeial method for the detection of mycoplasma is
available, but this culture method is lengthy and results can take a
number of days to complete. There are many alternative methods
such as PCR-based assays and biochemical assays measuring spe-
cific mycoplasma enzymes [35]. The PCR methods are becoming
increasing sophisticated and do provide a screen for many myco-
plasma species and can be used to identify specific species present.
Enzymatic methods are inexpensive and provide relatively
rapid turnaround of results, but can give inconclusive results.
It is advised that Pharmacopoeial (USP/EP) methods for ste-
rility are used. In general, these tests are often outsourced to spe-
cialist labs due to the large investment required to set up the
procedure in-house.
5.4 Prions The risks posed by transmissible spongiform encephalopathies

(TSEs) should be considered irrespective of the origin or history of
the cell line. There are a number of TSE diseases present across the
globe and their ability for transmission to humans, while unknown
in many cases, is plausible.
There exists a lack of clarity on the risks of prion diseases in cell
culture and variability in terms of uptake, persistence and expulsion
can be seen across different cell types. There are currently no quali-
fied assays that can be applied robustly in the context of cell cul-
tures; however, it is important to be aware of new developments in
this area where assays are being developed from those designed for
analysis of human tissues.
As well as the risks from cell sources it is important to also be
aware of the risks posed by reagents and raw materials used in cul-
ture. The use of low-risk reagents is highly advised in the context
of clinical applications [36].
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Chapter 7
Ethics and Governance of Stem Cell Banks

Donald Chalmers, Peter Rathjen, Joy Rathjen, and Dianne Nicol
Abstract
This chapter examines the ethical principles and governance frameworks for stem cell banks. Good gover-
nance of stem cell banks should balance facilitation of the clinical use of stem cells with the proper respect
and protection of stem cell sample providers and stem cell recipients and ensure compliance with national
regulatory requirements to foster public trust in the use of stem cell technology. Stem cell banks must
develop with regard to the science, the needs of scientists, and the requirements of the public, which will
benefit from this science. Given the international reach of this promising research and its clinical applica-
tion, it is necessary for stem cell bank governance frameworks to be harmonized across jurisdictions.
Key words Governance, Ethics, Stem cell banks, Harmonization, Standardization, Regulation,
Guardianship
1 Introduction
Stem cell science has accelerated since the isolation of pluripotent

stem cells from human embryos in 1998 and continues to attract
widespread international attention. Although the routine use stem
cells in medical practice is still some way off, a range of clinical
applications are already being trialled particularly in cancer treat-
ments [1]. The term “regenerative medicine,” coined in the 1990s,
is increasingly being used to refer to stem cell-based clinical appli-
cations. Basic stem cell science was marked by the traditional stan-
dards of self-regulation of science. Self-regulation has gradually
and increasingly moved toward established processes of standard-
ization of derivation and characterization of these cells. As the sci-
ence is becoming settled and standardized, scientists themselves,
research organizations, and also governments are establishing gov-
ernance infrastructures supporting this work [2], as preconditions
to clinical applications and eventual market products. In particular,
national and international efforts in stem cell research are being
matched by the establishment of stem cell banks and registries, as
essential repositories for transnational access to ethically sourced,
99
100 Donald Chalmers et al.
properly characterized and quality-controlled stem cell lines. Stem

cell scientists in the International Stem Cell Forum, International
Stem Cell Banking Initiative (ISCBI) have taken important steps
toward establishing “governance-by-standards” [3], in the science
and for stem cell banks in this dynamic, fast-moving, and technical
area. The need for standards and governance is incontestable as
translation of stem cell research to clinical applications develops,
particularly in countries, such as China [4] where clinics are offer-
ing “treatments” [5] that have not undergone proper testing in
approved clinical trials.
This chapter examines the ethical principles and governance
frameworks for stem cell banks. Good governance of stem cell
banks should balance facilitation of the clinical use of stem cells
with the proper respect and protection of stem cell sample provid-
ers and stem cell recipients and ensure compliance with national
regulatory requirements to foster public trust in the use of stem
cell technology. Stem cell banks must develop with regard to the
science, the needs of scientists, and the requirements of the public,
which will benefit from this science [6]. Given the international
reach of this promising research and its clinical application [7], it is
necessary for stem cell bank governance frameworks to be harmo-
nized across jurisdictions.
2 The Need for Stem Cell Banks
As the applications of stem cells move from the theoretical drawing

board to clinical applications, scientists have been considering their
requirements for stem cell sources. Stem cells to be used in clinical
and commercial applications will require strict standards for quality
control and documentation, as well as functional integrity. Early
targets for embryonic stem cell products, including retinal cells for
the treatment of Stagardt’s disease and development of cell-based
devices [8], tend to have been designed as “off the shelf” products.
These stem cell products have aimed at a wide customer base but
have required a single embryonic stem cell line for manufacture.
Similarly, early targets for the commercial development of mesen-
chymal stem cell-based products rely on the use of allogeneic cells
that can be harvested from donor material without consideration
of genetic matching or extraction and use of patients’ own stem
cells. These types of early target approaches have not required the
creation of stores, or banks, of many stem cell lines that can be
patient-specifically selected. When, and if, more patient-specific
approaches to regenerative medicine are developed, there will be
obvious requirements for banks of stem cells that can facilitate tis-
sue matching. At this stage, the development of private commer-
cial banks of cells is not eventuating.
To date, the development of stem cell banks is in the direction
of public banks in many jurisdictions. These public-sphere
Ethics and Governance of Stem Cell Banks 101
developments are being driven by scientists, research organiza-

tions, and governments. Publically funded enterprises, these stem
cell banks are conceptualized as essential resources for research and
emerging stem cell therapies, with clear missions to further stem
cell research and to act as repositories of multiple stem cell lines
that can be accessed and used globally. Although early in their
development, public stem cell banks hold the potential to become
repositories of clinical grade cell lines from a variety of genetic
backgrounds and of consistent and high-standard source materials
for use in clinical applications [9].
Stem cell banks should be distinguished from “cord blood
banks,” which are not discussed in this chapter. Cord blood refers
to residual umbilical cord and placental blood after a birth, which
contains stem cells that are frozen and may be later used in medical
applications [10]. Cord blood banks, unlike stem cell banks, are
being established generally within the private commercial sector to
provide private family storage for “peace of mind” [11] for parents
on the birth of their child. Cord blood banking offers long-term
storage of cells that may be seen as an “investment” [12] and
potentially available to treat the child’s diseases in the future. Cord
blood banks have been long established and the London Cord
Blood Bank as an example reached a 1000 collection in 1999 [13].
3 The Ethics of Stem Cell Derivation
Stem cells derived from human tissue, other than human embryos,
have generally not raised any controversy. This source is, however,
limited to providing adult stem cells of mature organs, populations
that are deficient in pluripotent stem cells and that cannot be
expanded efficiently in culture. As a consequence, these stem cells
are limited to research and clinical use and currently insufficient for
therapeutic development. Mesenchymal stem cells are an exception
and can be gathered from a number of body tissues, readily
expanded and are being explored as treatments for cartilage and
bone damage, inflammation and modulating the immune system
[14]. Transplanting such somatic stem cell populations has not
resulted in adverse safety indications, such as tumorgenesis.
Similarly, induced pluripotent stem cells (iPS cells) [15] have not
created controversy. This stem cell, created from the cells of the
adult, has the essential features of an embryonic stem cell, self-
renewal and pluripotency, or the ability to differentiate into all the
cell populations. iPS cell technology can provide an accessible
source of autologous patient-specific stem cells, thereby obviating
the need for immune matching or immune suppression, and a
source of disease-specific stem cells with applications in research
and drug development. Critically, the derivation of iPS cells [16]
does not require human embryos or donated oocytes.
In contrast, pluripotent embryonic stem cells derived from

early human embryos continue to raise controversy [17]. In 2012,
the National Institutes of Health (NIH) in the USA received pub-
lic submissions about the use of human embryonic stem cells and
resolved to suspend their use until these submissions were fully
considered [18]. The controversy continues to center on the philo-
sophic doubts about the moral status of the embryo [19, 20].
Embryo-derived stem cell promoted debates among established
churches and religions on the biological and moral definition of a
human embryo [21] and whether the early human embryo has the
status of a human being. Some countries legislated to prohibit
human embryo research, such as Austria, Ireland, Canada, and the
Philippines. In addition, the German Parliament prohibited
embryo research but, in 2002, with some ambivalence, allowed
embryonic stem cell research on imported lines [22, 23]. A broad
range of views on moral status of the embryo and research involv-
ing human embryos were expressed in the lengthy Australian
Parliament debates on the Research involving Human Embryos leg-
islation was debated in 2002 [24].
From a scientific, rather than an ethical, point of view, it is
generally accepted that there is fundamental research to be done
on comparisons between embryonic and adult stem cells and iPS
cells [25] to assess their utility in the development of safe cell ther-
apeutics for a range of diseases and in their application to drug
discovery and biomedical devices [26]. At this stage of scientific
development, it is not possible to advocate for the exclusive use of
one type of stem cell [27].
4 Principles for the Establishment, Good Governance, and Regulation

of Stem Cell Banks
With increasing acceptance of the research and clinical potential

and promise of stem cell science, there has been a movement
toward establishing repositories of stem cells. These repositories
have adopted the name “banks,” following the lead of human tis-
sue “biobanks.” The UK Stem Cell Bank, for example, was estab-
lished with the aim of providing a repository of human embryonic,
fetal, and adult stem cell lines. Its role is to provide an international
resource for stem cell research, supplying human stem cell lines,
both for research and to those wishing to develop cell lines for
clinical application [including] an active programme of precom-
petitive research in collaboration with academia and industry [28].
In addition, stem cell banks have also been established in countries
that are actively advancing the translation of stem cell science from
the bench to the clinic, particularly in the USA, Japan, Australia,
and Spain. In this respect, the work of the International Stem Cell
Forum, International Stem Cell Banking Initiative (ISCBI) of the

International Stem Cell Forum [29] aims to create a truly global
network of stem cell banks to facilitate best practice in stem cell
research and the eventual clinical application of these banked stem
cells. In this respect, the aims of the ISCBI align closely with those
of the International Society of Stem Cell Research (ISSCR) [1].
Stem cell banks are being established with effective and proper
organizational and governance structures [6]. With the increasing
prevalence of stem cell banks worldwide, it is timely to examine
core ethical principles and best practice governance frameworks
that should guide their establishment and use.
4.1 General Stem cell banks aim to support fundamental research and the trans-
Regulatory Framework lation of this research into effective clinical applications.
Accordingly, stem cell banks should establish governance struc-
tures appropriate for and consistent with these research and devel-
opment aims. Stem cell banks fall generally under the umbrella of
national legislation for human tissue and privacy legislation. Where
the stem cells are sourced from human embryos, there may exist a
further layer of national legislation or guidelines [30–34]. In some
countries, there is also specific human tissue legislation. The UK
has specific human tissue legislation [35] and, as the UK is a mem-
ber of the European Union, is a member of the European Union,
their stem cell bank is also governed by the EU Tissue and Cell
Directive [36], which sets standards for the quality and safety of
human tissue and cells for human applications.
Privacy regulation around the world is in broadly similar terms
and based on the OECD guidelines set in 1980. In Australia, as an
example, Privacy Principles require collection of data only for spec-
ified reasons; use and disclosure only for specified purpose; mainte-
nance of data quality; data security; rights of access and correction;
use of identifiers and nondisclosure; anonymity; limits on trans-
border data flows; and, special responsibilities for “sensitive infor-
mation,” which generally covers personal health information.
These principles are not dissimilar to most jurisdictions. Apart
from legislation, many countries have national research guidelines
for human tissue research and rules about the import and export of
tissue and cell lines [37]. Internationally, the Declaration of
Helsinki (1965) is the foundation for an essentially common inter-
national framework for research. The Declaration established the
key pillars for ethical review of medical research (voluntary consent
of the research participant; independent review of the project;
assessment of the risk; involvement of competent researchers of
integrity and research merit) [38]. These guidelines are contained
in some form of national code of ethical conduct in research. In
Australia, for example, the National Statement on Ethical Conduct
in Human Research 2007 sets down a comprehensive national
ethical regulatory framework for human research.
4.2 Responsibility Consistent with other branches of science, the stem cell scientific
of Stem Cell Scientists community has a responsibility to self-regulate and to consider and
and Governance assess the ethical and social dimensions of their science. The
Standards American National Academy of Science classically demonstrated
this principle of professional responsibility in science in 1975, at
the Asilomar Conference in 1975, when a precautionary approach
to biosafety of the new recombinant DNA research was adopted.
In a similar vein, Zelman Cowen, a former Governor-General of
Australia, recognized this social responsibility in saying that scien-
tists should be “... as active in [their] moral as in [their] scientific
imagination” [39].
Stem cell scientists have developed and promoted best practice
standards in their research [40]. The International Society for Stem
Cell Research (ISSCR) is an independent, nonprofit organization
established in 2002 to foster the exchange of information on stem
cell research and other collaborations amongst stem cell researchers
[41]. The ISSCR has issued Guidelines for the Clinical Translation
of Stem Cells, 2008 [42], which not only prescribe for the clinical
testing of stem cell therapies, consistent with national requirements,
but also address the major ethical issues surrounding stem cells [3].
In addition, in 2008, the International Stem Cell Banking Initiative
(ISCBI) was established to help develop a global network of stem
cell banks to exchange information about ethical and regulatory
issues and to develop a consensus statement on international mini-
mum standards for banking, characterization, and testing. The
ISCBI aims to establish a framework and “governance-by-standards”
[43], for international stem cell banking and research. The ISCBI
has published principles of best practice for the supply of human
embryonic stem cell lines for research purposes [44] and points to
consider for developing “Clinical Grade” pluripotent stem cell
[45]. These developments are essential as the translation of stem
cell research to clinical applications develops.
Through the ISSCR and the ISCBI, stem cell scientists are
responding ethically and responsibly to the challenges in the devel-
opment of this promising science.
4.3 Proper Detailed codes of governance and practice have been the hallmarks
Governance of the emerging stem cell banks. There has been uniform support
for the highest standards in governance and ethics in stem cell
banks to secure best practice in collection, deposit, storage, record-
keeping, access, distribution, use, tracking, and audit. These best
practices are aimed at ensuring that stem cell banks fulfill their twin
goals of maintenance of quality standards of deposited stem cells
(“depositors”) and equally high standards to those requesting use
the stem cells (“requestors”) for fundamental research or clinical
applications. In this respect, operators of stem cell banks should be
involved in the development and promotion of these standards and
should establish governance structures appropriate for and consis-
tent with these goals.
The UK Stem Cell Bank has an established governance framework

set out in its Code of Practice for the Use of Human Stem Cell Lines,
which “provides guidance on best practice for those working with stem
cell lines [and].. how the Steering Committee provides ethical over-
sight of UK research involving human embryonic stem cells” [46].
Importantly, this Code aims to “provide confidence and reassurance to
professionals and public alike that stem cell research in the UK is con-
ducted within a transparent and ethical framework.” Governance stan-
dards cover not only the internal management and administration
processes of the stem cell bank but also the public expectations for
transparency and accountability. Stem cell banks should include public
transparency procedures that allow public scrutiny and encourage pub-
lic trust. Public trust is an essential precondition for the successful oper-
ation and future research benefit of stem cell clinical applications
The Steering Committee of the UK Stem Cell Bank, under the
Code of Practice, receives reports from the Bank, which it oversees.
There is also a Management Committee, comprised of lay mem-
bers and representatives from research, healthcare, regulatory bod-
ies, and the Bank’s sponsors (including the Medical Research
Council). The Management Committee “monitors the Bank for
adherence to the Code of Practice and assists in the Bank’s strate-
gic development” [47].
A stem cell bank involved with research or providing stem cells
for research must comply with the established human research eth-
ics principles with independent ethical review of the research proj-
ect. In this regard, the integrity of a researcher is fundamental to
the ethical conduct of stem cell bank or other research.
4.4 Stem Cell Stem cell banks must ensure that the stem cells deposited have
Deposits been derived ethically and legally by the cell contributors. Like
and Guardianship other collections of banked human tissue, the governance struc-
of Stem Cell Material ture of a stem cell bank places guardianship responsibilities on the
bank to preserve and use the stem cells for the purposes established
for the bank [48]. In this respect, while there are many unique
ethical and legal aspects of stem cells, there are parallels between
stem cell banks and other tissue banks with respect to consent and
quality assurance. Deposits to stem cell banks require human ethics
agreements, which ensure that the cells have been obtained to an
ethical standard equivalent to the receiving country.
Deposits of stem cells into a bank should be voluntary and by
consent. Like a commercial trading bank, the “depositor” contin-
ues to own the stem cell samples that are held by the bank, which
can distribute them for research or clinical uses. The person who
wishes to use the stem cell lines must formally request the bank for
them and sign the relevant research use license [49]. Some stem
cell banks do not charge, except for freight costs for distribution of
cells but generally banks adopt some form of partial or full cost
recovery mechanism, often depending on whether the “requestor”
is from the commercial or academic sectors [50].
Stem cell line deposits are accompanied by a formal deposit

agreement. These material transfer agreements (MTAs) may have
other names. For example, the UK Stem Cell Bank receives depos-
its under their Material Deposit and Distribution Agreement
(MDDA). MTAs and MDDAs have the same purposes, namely to
transfer research materials and biological materials between two
organizations. They set out the duties of the stem cell bank but
also include the rights of the depositor for protection of commer-
cialization opportunities [51] and clarification of the arrangements
between the parties with respect to present and future downstream
intellectual property rights of the depositor [52]. Formalization is
particularly a necessary feature of stem cell MTAs because of the
need to clarify the quality and provenance of the stem cells. The
International Society for Biological and Environmental Repositories
(ISBER), in promoting uniform best practice standards for mate-
rial transfers, proposed standardization of common terms and the
types of considerations that MTAs “should address” [53]. The key
terms in stem cell MTAs generally specify: retention of ownership
of the cells by the depositor and return of materials; storage and
distribution of the cells by the bank; access to, use of, and restric-
tions on the distributed cells: intellectual property rights of the
depositor; confidentiality requirements; publication and attribu-
tion rights; liabilities and indemnities; and nonassignment of the
cells by the requestor. MTAs enable access to and release of stem
cells in banks and also enable records to be kept to trace the use of
the stem cells distributed under a research license. As stated, there
are clear parallels between stem cell banks and other tissue banks
and, clearly coding, recording, and traceability of stem cells are
essential [54]. All access to and release of information from stem
cell banks should be accompanied with proper and accurate records
of access to and release of cells or data. This traceable “chain of
responsibility” for every dealing in relation to the storage, han-
dling, and use is common to human tissue generally and is not
unique to stem cells. Stem cell bank MTAs will promote collabora-
tions between institutions to enable the development of the sci-
ence and clinical applications of stem cells.
Stem cell banks will be undertaking long-term storage of the
cells collected and the data. Banks should therefore ensure that
consent issues have been addressed and information provided to
those depositing cells. Information provided should include the
collection, use, storage, and transfer of stem cells or data, consis-
tent with the consent given. The stem cell bank governance struc-
ture should recognize the responsibilities for the deposited and
stored samples and the data derived. This idea of guardianship of
the cells has been described by the UK Biobank as acting “as the
steward of the resource, maintaining and building it for the public
good in accordance with its purpose” [55].
4.5 Quality The qualities of deposited stem cells, and the ability of deposited
Assurance and Safety cells to be used safely, are critical considerations for stem cell banks.
In comparison with adult stem cells, human embryonic stem cells
raise significant questions of safety. Embryonic stem cell lines can
accumulate genetic mutations and epigenetic modifications as a
consequence of continual expansion. These changes raise the pos-
sibility that lines could become refractive to differentiation, predis-
posed to tumor development, or exhibit other cellular malfunctions.
It is recognized that pluripotent cells can precipitate teratoma or
teratocarcinoma formation when introduced into the body, such
that the ability of the pluripotent cells to undergo complete dif-
ferentiation to a population that is no longer able to establish
tumors is considered an absolute requirement in the development
of cell therapeutics [56], and is an important manifestation of cell
quality for cells used in research and preclinical development. The
report by the National Academies Committee on the Biological
and Biomedical Application of Stem Cell Research noted that “[m]
ajor questions remain about the genetic and environmental fac-
tors…that control the fate of ES [embryonic stem] cells and about
the …various stages of cell differentiation” [57]. More recently,
the ISSCR Guidelines for the Clinical Translation of Stem Cells,
2008 [58], recognized the likely toxicity of stem cells and specifi-
cally recommended that: “Risks for tumorigenicity must be assessed
for any stem cell-based product, especially when extensively manip-
ulated in culture or when genetically modified. A clear plan to
assess the risks of tumorigenicity for any cell product must be
implemented under the direction of an independent review body
prior to approval for human clinical use.”
Quality assurance, or the ability to document stem cell sample
collection, deposit, storage, record-keeping, access, distribution,
use, tracking, and audit of processes, is projected as a key function
to be undertaken by banks to ensure deposited cell lines are not
tainted by human or process error. This requirement was a key
consideration when the National Institute for Biological Standards
and Control (NIBSC) was chosen originally as the host for UK
Stem Cell Bank. The NIBSC is a government-funded organization
involved in quality assurance and research related to product safety
and quality of biological medicines, which is a key consideration
for stem cell banks generally. The UK Stem Cell Line Registry dis-
tinguishes between “laboratory research grade” stem cell lines that
may only be used for research purposes and “clinical grade” lines
that have been approved by the relevant Regulatory Authority in
the source country, for clinical purposes [59]. Importantly, any
depositor of stem cell lines must immediately notify the Bank if
they become aware “of any property of the … line that has the
potential to significantly affect the quality, safety or efficacy of the
cells” [60]. This precautionary obligation is in line with the ISSCR
Guidelines for the Clinical Translation of Stem Cells 2008, which

provides helpful quality assurance recommendations on cell pro-
cessing, manufacture, and sourcing of stem cell material.
Importantly, these Guidelines are generally consistent with national
codes of ethical conduct in human research in requiring proper
preclinical studies to be conducted on animal models to assess the
efficacy of any proposed stem cell-based intervention. The ISSCR
Guidelines propose that any stem cell clinical application must be
carefully assessed for risk, properly communicated to the patient
and subject to independent scientific assessment and ethical review.
Also, potential benefits should not be overstated [61].
A study published in the journal Stem Cells noted that despite
the publicity on stem cell research, there is “scattered information
on the number of hESC [human embryonic stem cell] lines and the
degree, dynamics, and diversification of their use on a global level.”
The authors concluded that from just over one thousand original
human embryonic stem cell lines derived up to November 2009 (in
fact, 1071 at 87 institutions and in 24 countries) “only a fraction is
thoroughly characterized” [25]. It can be argued that quality assur-
ance extends beyond documentation of provenance and includes
validation of cells to meet basic functional requirements of pluripo-
tency and karyotypic integrity. It can be anticipated that as research
advances additional tests will be incorporated that may address other
cell qualities, including but not limited to epigenome integrity and
metabolic functions [62]. Similarly, as the understanding of human
embryonic stem cells and somatic stem cells advances exclusion cri-
teria for stored cell lines may be developed. The role of banks will
become critical in determining, in consultation with regulatory bod-
ies, best practice for the process of cell validation, ensuring the
appropriate application of validation practices, and acting as a reposi-
tory for cell information. The foreshadowed need for additional cell
validation raises the question, however, of how the maintenance of
cell compliance will be achieved and who is responsible for this task.
4.6 Avoiding The general principle of disclosure of interest is recognized in

Conflicts of Interest national codes for the responsible conduct of research. This gen-
eral principle applies to science and medical research journals,
which require declarations of conflicts generally and financial asso-
ciations with commercial organizations, in particular. This princi-
ple has extended to the emerging stem cell banks, which are
established to act as repositories for all types of well-characterized
and quality assured human stem cell lines for basic research and the
development of clinical applications. Interestingly, on the estab-
lishment of the UK Stem Cell Bank, it was an agreed principle that
the Bank’s research should avoid conflicts of interest with any basic
stem cell biology undertaken in the academic sector or market-
focused research in the commercial sector. The Bank’s research is
“restricted to areas relating to improving cell culture, preservation,
cell characterization and safety testing” [63].
4.7 Closure As a general principle, stem cell banks should have policies and
and Termination guidelines dealing with the possibility of closure of the bank and
of a Stem Cell Bank consequent transfer of the stem cells to another bank or institu-
tion. Any transfer should be determined by the terms of the deposit
and any agreements or conditions at the time of deposit.
4.8 International Stem cell banking is an international effort and, like other transna-
Harmonization tional research and development projects, requires collaboration
between participating institutions. Equally important, efforts need
to be made to harmonize the regulatory arrangements between
countries. It is unfortunate and unproductive if a researcher were
unable to secure specific stem cell lines because consent forms in
one country were not considered sufficient by a research ethics
committee in another country. It is highly desirable that interna-
tional best practice and ethical governance standards achieve, if not
uniformity, at least harmonization of standards. Stem cell banks
operate transnationally, so policies and guidelines should be in
place for conducting such transnational work [64]. There is an
ever-expanding library of international declarations, statements,
guidelines, and conventions encouraging international harmoniza-
tion of guidelines across jurisdictions. As an example, the UNESCO
International Declaration on Human Genetic Data (2003) requires
“the respect of human dignity and protection of human rights and
fundamental freedoms in the collection, processing, use and
storage of human genetic data, human proteomic data and of
…“biological samples,” in keeping with the requirements of equal-
ity, justice and solidarity” (Art 1a); and “consistent with the inter-
national law of human rights” (1b).
The International Stem Cell Banking Initiative (ISCBI) is pro-
moting a global network of stem cell banks committed to best
practice in research and clinical delivery. This objective includes
“the challenges for harmonizing stem cell banking on a global
level” [29].
5 Conclusion
Stem cells promise to introduce new and hopefully successful ther-

apies. The regulatory arrangements for stem cell banks should aim,
therefore, for the twin goals of research facilitation alongside assur-
ances of recipient protection. Public trust will be an imperative for
stem cell banks and is a fundamental cornerstone in the cultivation
and maintenance of trust in stem cell science and banking.
It is accepted that much basic research remains to be under-
taken before newly conceived stem cell therapeutics will be rou-
tinely used in the clinic. Stem cell banks have a vital role to play in
the development of stem cell research and applications. If devel-
oped to their full potential banks will enable the storage, docu-
mentation, and quality control of the best of those cell lines.
More importantly, through the potential to standardize ethical

and appropriate consent from donors, best-practice paradigms for
isolation of cells and tracking their provenance through MTAs, and
quality control through the development and adoption of widely
accepted and rigorous guidelines for cell assessment, banks have
the potential to be international drivers of quality and compliance
for human-derived stem cells into the future. Although the use of
banks by the research community is likely, uptake of banked cells
by commercial entities for the development of clinical applications
will depend on many factors, but most importantly the terms under
which commercial use of cells can be licensed, the quality of cells
that can be offered and the requirements placed on companies for
the quality assurance and validation of starting cell stocks. For
banks to achieve their stated goals they will need to be proactive,
occupying the interface between research and application and
ensuring the availability of cells that meet rigorous standards of
quality assurance, validation, and safety. Technical standardization
and self-regulation are as essential as the development of high stan-
dard governance arrangement for stem cell banks [2]. It is equally
important that stem cell banks are developed with due regard to
the needs of the scientists involved as and of the public that should
benefit from these developments. Finally, a strong cell banking
alliance will have a critical role to play in the globalization of cell
standards and cell compliance.
Acknowledgments
NHMRC Program Grant 490037; ARC Discovery Grant

DP11010069 for support.
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Part II
Protocols for Pluripotent Stem Cell Banking

Chapter 8
Derivation of Human Embryonic Stem Cells

Jeremy M. Crook, Lucy Kravets, Teija Peura, and Meri T. Firpo
Abstract
Embryonic stem cells (ESCs) represent a mainstay for pluripotent stem cell research and development
(R&D) and provide tangible opportunities for clinical translation including cell therapies and drug discov-
ery. Moreover, in spite of the discovery of induced pluripotent stem cells (iPSCs), ESCs are an essential
reference point, against which other pluripotent cells are compared. Hence, there is an ongoing need to
derive and bank quality-controlled research-grade and clinical-grade ESC lines using established and stan-
dardized methods. Here, we provide a concise, step-by-step protocol for the derivation of ESCs from
human embryos. While largely based on previously reported method for clinical-grade human ESC (hESC)
line derivation, the protocol is suitable for routine application, although adaptable for
clinical-compliance.
Key words Human embryonic stem cells, Blastocyst, Inner cell mass, Derivation
1 Introduction
Prior to the development of hESCs, the study of human embry-

onic development was performed primarily with animal models
and tumor cells with embryo-like characteristics. The use of hESCs
provides an alternative and arguably better approach to modeling,
since they are isolated directly from primary pluripotent cells of the
embryo inner cell mass (ICM). Notably, while an established hESC
line does not provide cells that are identical to their in vivo coun-
terparts, the cells maintain their capacity to self-renew and differ-
entiate into any cell type of the human body. These properties
render hESC line derivation valuable for both basic research and
clinical application.
The derivation of a single cell line consists of three primary
steps: (1) the culture of an embryo to blastocyst stage, (2) the
separation of the ICM from the trophectoderm outgrowth, and
(3) the establishment of the cell line by hESC expansion and char-
acterization. The resulting line comprises nonclonal pluripotent
cells of a single embryo.
115
116 Jeremy M. Crook et al.
The majority of embryo culture methods have been adapted

from protocols used by fertility clinics for in vitro fertilization
(IVF). There are several commercially available systems for the cul-
ture of human embryos, with older methods using in-house pre-
pared cryopreservants and media, with the latter commonly used
prior to the advent of regulator-compliant or -approved options. It
is therefore important to determine the approach taken by a clinic
or laboratory to culture and freeze embryos for hESC line deriva-
tion. Where possible, use of similar culture materials and methods
is advisable for consistency with procedure towards successful line
derivation. Notwithstanding, standard cell culture equipment and
reagents can be used to generate a hESC line, with a dissection
microscope helpful for embryo manipulation, and a phase-contrast
inverted microscope required to visualize the ICM outgrowth and
hESCs. While the following protocol is largely based on a previ-
ously reported method for clinical-grade hESC line derivation [1],
the protocol is suitable for routine application. In addition,
although the reagents and equipment are obtainable from a single
source, as standard components, similar items are available from
many cell culture and IVF suppliers. Several of these reagents and
suppliers are indicated in the notes section. Finally, the protocol
utilizes embryo bisection to separate the ICM from much of the
trophectoderm. Alternative methods are included or identified in
the notes as well.
2 Materials
2.1 Thawing 1. Cook Sydney IVF Blastocyst Medium.

of Embryos 2. Cook Sydney IVF Culture Oil (washed).
3. Cook Sydney IVF Thawing kit (including Cleavage thaw solu-
tions 1, 2, 3 and 4)* (see Note 1).
4. Cook Sydney IVF Blastocyst thawing kit (including Blastocyst
thaw solutions 1, 2, 3 and 4)* (see Note 2).
5. 35 mm diameter Petri dishes.
6. Nunc 4-well dish.
7. Sterile forceps.
8. Sterile scissors.
9. Spinal needle.
10. Transfer pipettes (0.29–0.31 mm, Vitrolife, Gothenburg,
Sweden).
11. Sterile water.
12. LN2 container.
13. P1000, P200 and P20 micropipettes.
Derivation of Human Embryonic Stem Cells 117
14. Automated pipettor.

15. 50 mL Sterile serological pipettes.
16. Stereomicroscope (Dissecting microscope).
17. Warm stage and pad for stereomicroscope.
18. Waterbath.
19. Thermometer.
20. Timer.
21. Benchtop incubator or plate warmer (MINC™ or Zander
IVF).
22. Incubator.
2.2 Bisecting 1. hESC medium. (DMEM base medium, Knockout Serum

and Plating of Human Replacer, Glutamine, Nonessential Amino Acids; all from Life
Embryos Technologies).
2. Irradiated mouse embryo fibroblast feeder cells.
3. Organ culture plate (Becton Dickinson).
4. bFGF-stock solution at 50 μg/mL (R&D Systems).
5. Cook Sydney IVF Cleavage/Blastocyst thaw solution 4.
7. T25 vented cap flasks.
9. Sterile Ultra Sharp Splitting Blade (Vigro ophthalmic blade).
10. Transfer pipettes (0.29–0.31 mm).
11. P1000, P200 and P20 micropipettes.
13. Stereomicroscope.
15. Alcohol burner.
16. Lighter.
17. Holder for denudation pipette.
18. Vitrolife rack for pipette holder.
19. Benchtop incubator or plate warmer.
20. Incubator.
2.3 Immunosurgery 1. Anti-human choriocarcinoma antiserum (anti-Bewo).

2. Guinea pig complement (Gibco-Invitrogen catalog
#19195-015).
3. DMEM (Gibco-Invitrogen catalog #11965-092).
4. Fetal bovine serum, characterized (Hyclone SH30071.03).
5. Petri dishes.
6. Pulled Pasteur pipette with the tip diameter ~30 μ or the inner
diameter equal to the size of the ICM.
7. Micrometer pipette.
8. hESC medium. (DMEM base medium, Knockout Serum
Replacer, Glutamine, Nonessential Amino Acids; all from Life
Technologies).
12. Cook Sydney IVF Cleavage/Blastocyst thaw solution 4.
16. Transfer pipettes (0.29–0.31 mm.)
17. P1000, P200, and P20 micropipettes.
18. Automated pipetman.
21. Alcohol burner.
22. Lighter.
26. Incubator.
2.4 Preparation 1. hESC medium. (DMEM base medium, Knockout Serum

of Plates for Culture Replacer, Glutamine, Nonessential Amino Acids; all from Life
of Human Embryos Technologies).
8. Sterile Ultra Sharp Splitting Blade (Vigro ophthalmic blade).
14. Alcohol burner.
15. Lighter.
19. Incubator.
2.5 Passaging 1. T25 vented cap flasks.

of Embryonic 2. 2 mL Sterile serological pipettes.
Outgrowths for hESC
Line Derivation
4. hESC basal medium.
5. bFGF-stock solution at 50 μg/mL
6. Organ culture plate with feeders.
7. Sterile Ultra Sharp Splitting Blade.
8. Stem cell cutting tool (0.29–0.31 mm).
14. Alcohol burner or low Bunsen burner.
15. Lighter.
19. Cell culture incubator.
3 Methods
3.1 Thawing As the first step in the process of deriving a hESC line, thawing an
of Embryos embryo requires consideration of the cryopreservation method
used by the provider (e.g. IVF clinic). In addition, the quality of
the embryo, stage of development, and duration of storage can
affect the success of thawing. Historically, clinics have used in-
house cryopreservants and applied variants of several published
protocols for freezing and thawing. More recently, FDA-approved
and CE-marked kits have become widely available, with separate
kits used for thawing embryos from the cleavage and blastocyst
stages. Note also the specific instructions applicable to embryos

cryopreserved in straws using slow-freezing technique with a pro-
grammable controlled-rate freezer. For vitrified embryos, the
warming instructions provided by the vitrification system supplier
should be used to the point of removal of embryos from the warm-
ing solutions. Notwithstanding, thawing generally comprises three
steps: (1) the evaporation of LN2 at room temperature (RT), (2)
the rapid thaw of the straw contents (usually at 30–35 °C), and the
systematic dilution of the cryopreservation medium in growth
medium. A traditional approach would use the same system for the
freeze and thaw, but many laboratories now use a kit specifically
developed for thawing.
If the thaw is successful, the embryo should appear to be at the
same stage of development as on the day of freezing based on size,
clarity, and granularity of the blastomere cytoplasm, and without
dead blastomeres. Embryos must be cultured in growth medium
that is appropriate for the stage of development until reaching blas-
tocyst stage. Embryos thawed at the blastocyst stage may appear to
be collapsed, but should reinflate within a few hours to regenerate
typical blastocyst morphology. At this time, ICM may be clearly
visible. If not, rotation of the embryo may help to visualize the
ICM from a different angle. In addition, human ICM, unlike
mouse ICM, may spread out as a thin layer that adheres close to
the surrounding trophectoderm, and therefore may not be
apparent.
While here, we recommend the Cook Sydney IVF medium
and thawing kit, other kits and medium are available from Vitrolife,
Cooper Surgical (Quinn), Sage, or Irvine Scientific (see Note 3). If
possible, we also advocate consultation with the embryologist
responsible for generating embryos used for line derivation.
1. Pipet 700 μL of Cook Sydney IVF Blastocyst medium and
300 μL Cook Sydney IVF Culture Oil into well #1 of a Nunc
4-well dish (see Note 4), and two 10 μL drops of Cook Sydney
IVF Blastocyst medium and 700 μL of Cook Sydney IVF
Culture Oil on each of wells #2 and #3 (see Note 5).
2. Equilibrate overnight in a humidified incubator at 37 °C, 5%
CO2.
3. Remove the thaw solutions from the refrigerator to RT at least
30 min prior thawing. Determine which thawing kit to use
based on the stage of the embryo.
(a) If thawing Day 3 embryos (cleavage stage): Use Cook
Sydney IVF Thawing kit Cleavage thaw solutions 1, 2, 3
and 4.
(b) If thawing Day 5–6 embryos: Use Cook Sydney IVF
Thawing kit: Blastocyst thaw solutions 1, 2, 3 and 4.
Table 1
Configuration of thaw solutions in 4-well dish
Well number Solution

Well 1 0.5 mL of thaw solution #1
3. Pipet the solutions into a Nunc 4-well dish as indicated in the

Table 1.
4. Transfer two 35 mm Petri dishes to the biosafety cabinet
(BSC). Leave all dishes to warm at RT for at least 30 min
before adding the solutions. If required, pipet 1 mL of Thaw
Solution 1 into one Petri dish.
5. Heat the water bath to 30 °C.
6. Remove the straw from the LN2 container with sterile forceps
(see Notes 6 and 7).
7. Hold straw at RT for 30 s.
8. Immerse the straw in the 30 °C water bath for 30–40 s.
9. Wipe dry the outside of the straw with a sterile lint-free
tissue.
10. Using sterile scissors, cut off the end of the straw sealed with
the putty. Some clinics use alternate vessel types, such as glass
vials or tubes, or other sealing methods that may require spe-
cial methods for opening (see Note 7).
11. Using a sterile spinal needle, push the cotton plug down the
straw until all liquid (including the embryo) is expelled from
the straw into an empty Petri dish.
12. Record the time of expulsion (see Note 8).
13. Transfer the embryo with a transfer pipette into well #1 of the
Nunc 4-well dish (refer to step 4). Record time of transfer (see
Note 9).
14. Transfer the embryo through the following incubation steps:
(a) Cleavage (or Blastocyst) Thaw 1–10 min at RT.
(b) Cleavage (or Blastocyst) Thaw 2–10 min at RT.
(c) Cleavage (or Blastocyst) Thaw 3–10 min at RT.
(d) Cleavage (or Blastocyst) Thaw 4–10 min at RT followed
by 10 min at 37 °C using warm pad
15. Transfer a Nunc 4-well dish from step 1 to the BSC. Transfer
embryo with a transfer pipette to pre-equilibrated culture
Table 2
Embryo assessment
Date and time Hours after thawing Embryo quality assessment
medium in well #1, then into a 10 μL drop of Cook Sydney

IVF Blastocyst medium. Assess the quality of embryo.
16. Transfer the dish into incubator and incubate at 37 °C in a
humidified incubator at 5% CO2.
17. Reassess embryo (see Notes 10–13; Table 2).
18. Conclude embryo culture (see Note 14).
3.2 Preparing Plates 1. Thaw reagents.

for ICM Culture 2. Assemble Stem Cell Basal Medium (500 mL):
3.2.1 Medium (a) DMEM base medium 400 mL
Preparation (b) Knockout Serum Replacer 100 mL (final concentration
20%)
(c) Glutamine 5 mL of 200 mM stock (final concentration
1 mM)
(d) Nonessential Amino Acids 5 mL of 10 mM stock (final
concentration 0.1 mM)
(e) β-Mercaptoethanol 3.5 μL of 14.3 M stock (final concen-
tration 0.1 mM)
3. Assemble Stem Cell/FGF Medium (10 0 mL)
(a) Stem Cell Basal Medium 100 mL
(b) FGF-2 0.5 ml of 10 μg/mL stock (final concentration
50 ng/mL) (see Note 15).
3.3 Feeder Plate 1. To 500 mL of water for embryo transfer (Sigma catalog
Preparation #W1503), add 0.5 g gelatin (Sigma G1890). Swirl and then
put the bottle in a 37 °C water bath until crystals are com-
pletely dissolved. Filter through 0.22 μ filter and store
refrigerated.
2. Transfer gelatin solution to tissue culture plates, dishes or
flasks. Use 8–20 mL for a T175 flask, 2–5 mL for a 10 cm dish,
0.5–2 mL for a 60 mm dish or 6-well plate well, 200–500 μL
per well of 24-well plate or 4-well plate. Swirl to wet the entire
surface of the dish. Coat the next dish and repeat until all
required dishes are coated. Use gelatin coated culture vessels
immediately.
3. Remove vial of feeder cells from LN2 freezer.
4. Place vial in 37 °C waterbath until ice is completely thawed.
5. Add feeder medium to vial (2–5 mL for vial frozen from a

single confluent T175 flask).
6. Transfer cell suspension to centrifuge tube.
7. Centrifuge for 3–8 min at 300 × g.
8. Aspirate the supernatant, and resuspend the cells in feeder
medium.
9. Plate in gelatinized tissue culture plates.
10. Incubate until needed, no longer than 1 week before use as
feeders.
3.4 Bisecting Bisection allows the removal of the zona pellucida from the embryo
and Plating of Human to allow it to adhere to feeders under the following culture steps.
Embryos As an alternative, the zona pellucida can be removed by protease
digestion, or by acid tyrode treatment (see Note 16). Further, the
bisection serves to remove much of the trophectoderm from the
ICM of the embryo, reducing the risk of trophectoderm over-
growth in the stem cell culture. Trophectoderm can also be
removed by dissection with a micromanipulator, or by complement-
mediated cell lysis (immunosurgery: see Subheading 3.5).
Once the ICM has been isolated, it is transferred to hESC cul-
ture conditions. These consist of an appropriate medium and a
layer of extracellular matrix (ECM), provided by, for example, a
feeder layer of inactivated mouse or human embryo fibroblasts.
The feeder layer can be replaced by isolated ECM, such as
Matrigel™ [2, 3], or by specific matrix components, such as vitro-
nectin [4, 5]. If matrix components are substituted for the feeders,
bFGF concentrations should be increased to 100 ng/mL [6].
Many hESC media are available commercially and/or detailed in
publications, most of which contain 4–50 ng/mL of bFGF for
hESCs derived or cultured on feeders, and 100 ng/mL for those
with purified matrix components [6, 7].
1. Remove the prepared hESC medium from the refrigerator to
the incubator 1 day before embryo bisection and plating for
equilibration. Add up to 70 mL of the completed hESC
medium to T25 flask.
2. Place T25 flask containing medium into a humidified incuba-
tor at 37 °C, 5% CO2 overnight.
3. Next day assess embryo to confirm that is suitable for plating
under the microscope.
4. Criteria for the assessment are:
(a) Embryo survived thawing
(b) Blastocyst stage embryos re-expanded
(c) Most of the blastomeres are intact and compacted, not lysed
(d) Embryo looks viable (see Note 17)
5. Remove Cook Sydney IVF Cleavage/Blastocyst Thawing

Solution 4 from the refrigerator.
6. Pipet 3 mL of the thaw solution 4 into a Petri dish that will be
used for embryo bisection.
7. Incubate the dish containing the thaw solution on the micro-
scope warm stage for at least 15 min.
8. Add required/calculated amount of bFGF stock to T-25 flask
containing hESC medium (prepared the day before) to make a
final concentration at 50 ng/mL (see Note 16).
9. Observe by microscope the organ culture plate with feeders
and ensure that it is suitable to receive the bisected embryo (see
Note 18).
10. Aspirate the feeder medium and add 1 mL of bFGF-
supplemented hESC medium into the human feeder organ
culture plate.
11. Return the organ culture plate with feeders to the incubator.
12. Attach the embryo transfer pipette to the holder for denuda-
tion pipette. Light the alcohol burner, smooth the end of the
transfer pipette in a flame, and then let it rest on a pipette
holder rack.
13. Transfer the embryo culture plate from the incubator to the
BSC.
14. Transfer the embryo to the bisection dish (refer to step 6).
15. Under the stereomicroscope, bisect the embryo into two parts
with a sterile ultra-sharp splitting blade, leaving the intact ICM
with one part.
16. Using a blade and/or a transfer pipette, remove any remnants
of the zona pellucida from the embryo halves.
17. Transfer the part with the ICM to the human feeder organ
culture plate and place the plate in the incubator.
18. Discard the other half of the embryo with the trophectodermal
cells (see Notes 18 and 19).
3.5 Alternative 1. Prepare 2× Petri dishes with 8× drops each of serum-contain-

Method to Bisection: ing medium for washing.
Immunosurgery 2. Dilute anti-human antibody 1:10 with serum-free medium
(30–50 μL final volume).
3. Put 2 × 20 μL drops of diluted antibody into another Petri
dish.
4. Remove embryo(s) from culture using a pulled Pasteur pipette
and put in one of the antibody drops.
5. Transfer the embryo(s) to the second antibody drop with
minimum medium carryover and leave at RT for 25–35 min.
6. Wash embryo: First, pick up the embryo from the antibody

drop and deposit into the first of the eight wash drops in one
of the dishes. Then, successively transfer the embryo through
the remaining drops until the eighth drop.
7. Dilute complement 1:10 with serum-free medium (30–50 μL
total volume).
8. Put diluted complement in two drops in a Petri dish and trans-
fer washed embryo to the first drop.
9. Transfer the embryo(s) to the second drop with minimal car-
ryover of medium, and leave the dish at RT for 20–30 min.
Observe for trophoblast cell lysis as evidenced by blistering or
blebbing after approximately 20 min.
10. After observing lysis of trophectoderm cells, wash embryo(s)
with eight transfers of serum-containing wash medium drops
in the second dish.
11. In the eighth drop, pipet embryos (one at a time) in and out of
the pulled Pasteur pipette with tip diameter about half the
diameter of the blastocyst. Lysed trophoblast cells should
come off, leaving a clump of intact cells, the ICM.
12. Transfer the ICM to the feeder-containing well (4-well plate or
center well of organ culture plate) for hESC line derivation.
3.6 Passaging 1. Assess the quality and development of embryo outgrowth (see
of Embryonic Notes 13 and 14). Record in the Table 3.
Outgrowths for hESC 2. Remove the completed hESC medium from the refrigerator to
Derivation the BSC 1 day before embryo bisection and plating. Add up to
70 mL of the completed hESC medium to T25 flask.
3. Place T25 flask containing medium in a humidified incubator
overnight at 37 °C, 5% CO2.
4. Remove bFGF stock solution from the freezer and pre-
equilibrated hESC medium from the incubator.
5. Add required/calculated amount of bFGF stock to T-25 flask
containing hESC medium (prepared 1 day before) to make a
final concentration at 50 ng/mL (see Note 16).
Table 3
Quality and development of embryo outgrowth
Outgrowth
Date and time Days after plating quality plating
Estimated date for the next passage
Putative hECS colony observed N
days after embryo plating
6. Observe by microscope the organ culture plate with feeders

and ensure that is in suitable condition to receive the embryo
outgrowths (see Note 20).
7. Aspirate the feeder medium. Add 1 mL of bFGF supplemented
hESC medium into organ culture plate with feeders.
8. Return the organ culture plate with feeders to the incubator.
9. Attach the embryo transfer pipette to the holder for denuda-
tion pipette. Light the alcohol burner and smooth the end of
the transfer pipette in a flame, then let it rest on a pipette
holder rack.
10. Transfer the embryo outgrowth plate and the human feeder
organ culture plate from step 6 from incubator to BSC.
11. Under the stereomicroscope, using either a sterile ultra-sharp
splitting blade or a stem cell cutting tool, cut the outgrowth
into two to eight pieces, depending on the size of the
outgrowth.
12. Using a sterile transfer pipette, transfer the fragment(s) to a
human feeder organ culture plate and return the newly pas-
saged outgrowth plate(s) to the incubator.
13. Record details of passaging activity such as:
(a) Passage Number
(b) Number of fragments
(c) Appearance of outgrowths
14. Change the medium of newly passaged plates daily (see Note 21).
15. Continue culture of putative hESC lines (see Notes 22–24).
4 Notes
1. The thawing of embryos for hESC line derivation is the same

for Day 3 or Day 5–6 cleavage stage embryos. However, differ-
ent embryos will use different media, and require different cul-
ture time for reassessment of the embryo before going into
bisecting and plating of the blastocyst.
2. Items marked with * are thawing solutions, specific for the
stage of embryo, with only one to be used in each procedure.
3. The following are resources for (a) cleavage stage and (b) blas-
tocyst stage embryos from:
(a) Cook Medical: 95 Brandl Street, Eight Mile Plains,
Brisbane, QLD 4113, Australia: (a) Sydney IVF thawing
kit (b) Sydney IVF Blastocyst Thawing Kit http://www.
cookmedical.com
(b) Vitrolife, Inc.: 3601 South Inca Street, Englewood, CO

80110 (a) Thaw kit 1 (b) G-thaw kit Blast http://www.
vitrolife.com
(c) Sage: While this company provides separate media for cul-
turing cleavage stage and blastocyst stage embryos, they
offer a single thaw kit for both stages.
(d) Cooper Surgical: 75 Corporate Drive Trumbull, CT 06611
USA 95 Corporate Drive, Trumbull, CT 06611 (a) and (b)
Quinn’s Advantage Media http://www.coopersurgical.com
(e) Irvine Scientific: 2511 Daimler Street Santa Ana, CA
92705-5588 (a) Embryo Thaw Media Kit. (b) Blastocyst
Thaw Media Kit http://www.irvinesci.com/
4. As an alternative to 4-well plates, individual Falcon organ cul-
ture plates or 60 mm plates can be used (Becton-Dickinson 1
Becton Drive, Franklin Lakes, NJ USA 07417 http://catalog.
bd.com). For 60 mm plates, medium can be placed as droplets
with a sterile mineral oil overlay to prevent evaporation.
5. Because the volume of medium is small, and osmolarity is an
important factor in embryo culture, clinical-grade mineral oil
is commonly used. The oil is gas permeable. Mineral oil can be
obtained from the sources listed (see Note 3).
6. Wear safety goggles, cryogenic apron, and cryogenic gloves
when handling LN2 before the actual thawing procedure.
Handle LN2 in a well-ventilated room.
7. Prior to the use of plastic straws, glass straws and sealed vials
were commonly employed for embryo cryopreservation. To
avoid injury and premature thawing of embryos, exceptional
care must be taken in handling glass vials. To open a glass vial,
first fill a wide-mouthed dewar flask with LN2. Then, hold the
vial (covered with sterile gauze) in the gas phase of LN2 over
the wide-mouthed dewar flask and carefully break the glass at
the score.
8. It is helpful to observe the embryo as it exits the straw using a
microscope to be sure it has been retrieved. The same applies
for handling the embryo by transfer pipette in the following
steps.
9. It is important to transfer as little fluid with the embryo as pos-
sible during serial transfer through dilute cryopreservant and
replacement with culture medium.
10. Reassess the quality of Day 3 embryos at least every 24–48 h.
Culture the embryos 48–49 h before bisecting and plating.
11. Reassess the quality of Day 5–6 embryos at least every 24 h.
Culture the embryos for 4–48 h before bisecting and plating.
12. The exact time of culture before bisection and plating of

embryos is based on the operator’s judgment of embryo stage
and quality.
13. Assess each culture based on the following criteria:
(a) Embryo has survived the thawing procedure.
(b) Most of the blastomeres are intact and compacted (not
lysed).
14. An embryo culture must be terminated when the quality of the
embryo has deteriorated to an extent that no cell line can be
obtained. Termination of culture should be based on the oper-
ator’s judgment.
15. FGF-2 should be added immediately prior to use.
16. Acid tyrode kits can be purchased from Irvine Scientific and
Cooper Surgical Protease solutions can be purchased from
Vitrolife.
17. The assessment of viability is not necessarily the same as when
assessing suitability for embryo transfer, as embryos with poor
prognosis for establishment of pregnancy can still yield viable
stem cell lines.
18. If in doubt about the success of bisection, both halves can be
plated onto the culture plate.
19. If the embryo is deemed unsuitable for bisection, it should be
plated whole. Only the zona pellucida (if still surrounding the
embryo) is cut open with an ultra-sharp splitting blade to
release the embryo, which is then transferred to the feeder
organ culture plate.
20. Following plating, inactivated human feeder cells are able to
support undifferentiated hESC growth for up to 3 weeks.
However, they should be used within 7 days after plating.
21. Changing medium: tip plate at an angle, and aspirate medium
with p1000 pipette or with Pasteur pipette with suction. Gently
replace medium on the side of the plate to prevent peeling of
feeders or embryo.
22. Using the stereomicroscope or inverted microscope with
phase-contrast optics, assess the plated embryonic outgrowth
daily or every other day, starting 1–2 days after plating. Record
the status of outgrowths.
23. The timing of the first few passages of the embryonic out-
growths is dependent on the operator’s judgment. Only well-
defined outgrowths have to be passaged (likely between 4 and
8 days from plating). Criteria are:
(a) Visible layer of cells emanating from the original embryo/
ICM is seen attached to the feeders, but clearly distin-
guishable from the feeder cells by appearance.
(b) Cells possibly also consisting of a defined, round ICM

colony.
(c) If no well-defined outgrowth is observed by Day 11 from
embryo plating, the outgrowth is passaged.
(d) Depending on operator’s judgment, the old plate can be
left to continue in culture for additional 3–10 days.
24. If no new outgrowths are observed, the plate is to be discarded,
timing based on the operator’s judgment, or by 14 days in
culture.
(a) Culture of the outgrowth must be terminated if after a few
passages the outgrowth degrades beyond recovery to an
extent that a cell line cannot be obtained.
(b) If a new outgrowth is observed, it must be passaged and
combined with the highest passage plate of the same out-
growth/hECS line.
Acknowledgment
J.M.C. acknowledges funding from the Australian Research

Council (ARC) Centre of Excellence Scheme (CE140100012).
References
1. Crook JM, Peura TT, Kravets L et al (2007) cells in defined conditions. Nat Biotechnol
The generation of six clinical grade human 2:185–187
embryonic stem cell lines. Cell Stem Cell 5. Vemuri MC, Schimmel T, Colls P et al (2007)
1:490–494 Derivation of human embryonic stem cells in xeno-
2. Xu C, Inokuma MS, Denham J et al (2001) free conditions. Methods Mol Biol 407:1–10
Feeder-free growth of undifferentiated 6. Xu RH, Peck RM, Li DS et al (2005) Basic FGF
human embryonic stem cells. Nat Biotechnol and suppression of BMP signaling sustain undif-
10:971–974 ferentiated proliferation of human ES cells. Nat
3. McWhir J, Wojtacha D, Thomson A (2006) Methods 3:185–190
Routine culture and differentiation of human 7. Meng G, Liu S, Rancourt DE (2012) Synergistic
embryonic stem cells. Methods Mol Biol effect of medium, matrix, and exogenous factors
331:77–90 on the adhesion and growth of human pluripo-
4. Ludwig TE, Levenstein ME, Jones JM et al tent stem cells under defined, xeno-free condi-
(2006) Derivation of human embryonic stem tions. Stem Cells Dev 11:2036–2048
Chapter 9
Derivation of Human-Induced Pluripotent Stem Cells

in Chemically Defined Medium
Guokai Chen and Mahendra Rao
Abstract
Human somatic cells can be reprogrammed by defined factors to induced pluripotent stem cells (iPSCs).
Importantly, the quality of iPSCs could impact the potential of these cells in basic and clinic research.
Here, we describe a method to reprogram human fibroblast cells with Sendai virus in chemically defined
conditions, to generate iPSCs that are integration-free and suitable for research and translational
applications.
Key words Induced pluripotent stem cells, Reprogramming, Chemically defined, Sendai virus
1 Introduction
Nuclear reprogramming by defined factors revolutionized pluripo-

tent stem cell research, and researchers are now able to easily get
access to patient-specific iPSCs [1–3]. These iPSCs have potential
to be used for drug screening, disease modeling, or as starting
materials for cell-based therapies. In the past 5 years, different
somatic cell types have been successfully reprogrammed; however,
the reprogramming efficiency can vary significantly due to different
combinations of defined factors and small chemicals used in the
process. At the same time, most reprogramming experiments are
conducted on feeder cells with undefined culture medium, where
batch-to-batch variation of the reagents often greatly affects the
quality of iPSCs obtained in such conditions. Fibroblast cells are
one of the most popular somatic sources for reprograming. Here,
we describe a simple method to effectively and consistently repro-
gram fibroblast cells in chemically defined medium independent of
feeder cells [4]. We use the Sendai virus approach as an example to
demonstrate the fibroblast reprogramming process. Sendai virus is
a RNA virus that does not integrate into the host genome, so it can
help derive iPSCs without integration [5–7]. The reprogramming
131
132 Guokai Chen and Mahendra Rao
Fig. 1 iPSC derivation procedure by Sendai virus in defined culture. (a) Major steps in the derivation process.
(b) Growth medium change during iPSC derivation
procedure can easily be adapted to other reprogramming approaches

such as lentivirus, retrovirus, and episomal plasmid DNA [1, 3, 8].
In this protocol somatic cells are reprogrammed in chemically
defined medium, which significantly improves the consistency and
efficiency (Fig. 1). At the same time, the chemically defined condi-
tions used here can be easily modified to meet the Good
Manufacturing Practice (GMP) standard, which could have great
application potential in translational research. When the optimal
integration method is finally established, this procedure can be eas-
ily adapted to generate iPSCs suitable for translational applications
(Fig. 2).
2 Materials
2.1 Cells 1. Human fibroblasts.
2.2 Media 1. For the medium change schedule, refer to Fig. 1b.
2. Reprogramming medium 1: DMEM/F12 (Invitrogen),
64 mg/L L-Ascorbic acid 2-phosphate magnesium salt, 14 μg/L
Sodium Selenite, 10.7 mg/L Holo-transferrin, 100 μg/L
basic FGF, 20 mg/L Insulin, and 1 μM Hydrocortisone.
Adjust to pH 7.4 with HCl or NaOH, and then adjust medium
to 340 mOsm/L osmolarity (see Notes 1 and 2).
Derivation of Human-Induced Pluripotent Stem Cells in Chemically Defined Medium 133
Fig. 2 Cell morphology during iPSC derivation and characterization by FACS. (a) Cell morphology changes from
fibroblast to iPSC colony. (b) Flow cytometry profile of expanded iPSCs
3. Reprogramming medium 2: DMEM/F12 (Invitrogen),

Sodium Selenite, 10.7 mg/L Holo-transferrin, 100 μg/L basic
FGF, 20 mg/L Insulin. 100 μM Sodium Butyrate is often
added to promote reprogramming efficiency. Adjust to pH 7.4
with 340 mOsm/L osmolarity (see Notes 1 and 3).
4. Human ESC/iPSC E8 medium: DMEM/F12 (Invitrogen),
Sodium Selenite, 10.7 mg/L Holo-transferrin, 100 μg/L basic
FGF, 1.8 μg/L TGFβ1, 20 mg/L Insulin. Adjust to pH 7.4
with 340 mOsm/L osmolarity (see Notes 1 and 4).
5. Fibroblast medium: 10% Fetal Bovine Serum (Hyclone) in
DMEM, 1× Nonessential amino acid, store at 4 °C (see Note 5).
2.3 Passaging 1. Fibroblast dissociation: TryPLE (Life Technology) (see Note 6).
2. ESC/iPSC dissociation: EDTA (see Note 7).
2.4 Reprogramming 1. CytoTune-iPS Sendai Reprogramming Kit (Life Technology,

Catalog number 1378001). The kit contains four viruses,
respectively overexpressing OCT3/4, SOX2, KLF4, and
cMYC genes (see Note 8).
2.5 Categorization 1. APS Staining.

3 Methods
3.1 Fibroblast 1. Day 0—Take low-passage fibroblast culture and plate in 1 well
Reprogramming with of a 6-well dish so that cells will be ~80% confluent the next
Sendai Virus (Fig. 1a, b) day (100,000 or 150,000 cells/well) in fibroblast media
(see Note 5).
2. Day 1—Thaw the four vials of Sendai virus on ice and com-
bine in one tube.
3. Still Day 1—Aliquot 100 μL Sendai virus into the well con-
taining fibroblasts. Mix the virus well in the medium by pipet-
ting. Incubate cells with the virus at 37 °C (see Note 9).
4. Day 2—Use 12 mL ice-cold or 4 °C DMEM/F12 medium to
resuspend frozen 2 mg Matrigel®, and aliquot the mixture
into two 6-well plates (1 mL/plate). Incubate the plates for at
least 30 min at 37 °C or room temperature (RT; see Notes 9
and 10).
5. Still Day 2—Briefly wash the fibroblasts with PBS; treat the
cells with 1 mL TrypLE™ at 37 °C for 5 min; neutralize and
wash off the cells with 5 mL Reprogramming medium 1 (see
Notes 6 and 11).
6. Spin the cells down at 200 × g for 5 min. Spin cells down and
resuspend them in Reprogramming medium 1. Plate cells
onto Matrigel® coated plates (1 well of infected cells into 2 × 6
well plates coated with Matrigel®), also in Reprogramming
medium 1 (see Notes 2 and 12).
7. Keep cells in Reprogramming medium 1, and feed them every
other day for 3–5 days, until ~40–50% confluency (see Note 13).
8. Day 5 or 7 (approximately)—Change medium to Reprogram-
ming medium 2. 100 μM Sodium Butyrate is often added to at
least 3 wells to promote reprogramming efficiency (see Note 14).
9. Continue feeding every other day (see Notes 15 and 16).
10. Around Day 20–25, colonies should be ready to try picking.
Around this time original reprogramming plates should receive
normal E8 media (with TGFβ1) daily, if they have not been
switched to this already (see Note 17).
11. For picking—Prep a 24-well plate by coating with Matrigel®.
After coating, add E8 (TGFβ1 media) with 1× ROCK inhibi-
tor added to each well (see Note 18).
12. Find colonies under microscope with 4× objective. Dislocate
colonies with 20 p or 200 μL pipette and transfer the colonies
into individual wells (see Note 19).
13. Transfer colony pieces from a single colony into 1 well of a
24-well plate. Repeat with other colonies until desired num-
ber of colonies/wells is plated. The day after picking, change
media to E8 (TGFβ1) without ROCK inhibitor. Feed daily

until colony is big enough to passage (see Note 19).
14. When a colony is ready to passage, use EDTA to passage indi-
vidual colonies. Put a large portion of the cells into a Matrigel®
coated well on a 12 or 6-well plate, while aliquoting a small
portion into one well of a Matrigel®-coated 24 well (with 1×
ROCK inhibitor) (see Note 20).
15. After a few days, use APS staining to confirm the pluripotency
of the clones. Retain the positive clones for further expansion
and cryopreservation.
16. Once expanded, the iPSCs can then be characterized as
appropriate.
4 Notes
1. Chemically defined media only contains essential components,

and growth factors are often not stable at 37 °C, so the media
used here should be always stored at 4 °C. We recommend
that you do not warm medium using a 37 °C water bath. Cool
medium can be directly used to feed the cells.
2. Reprogramming medium 1 is suitable for short-term fibro-
blast growth, and can facilitate fibroblast reprogramming.
However, fibroblast overgrowth could inhibit iPSC expansion
in later stages, so medium needs to be switched to
Reprogramming medium 2.
3. Reprogramming medium 2 is suitable for iPSC clonal expan-
sion, and fibroblast cells proliferate slower in it, when com-
pared to Reprogramming medium 1. Sodium Butyrate is an
efficient small chemical that improves reprogramming effi-
ciency in this culture condition.
4. Human ESC/iPSC E8 medium is specifically designed to
maintain human ESC and iPSCs. This medium can be used to
maintain iPSCs when the cells become over-confluent after
2–3 weeks during reprogramming, and it is also used to main-
tain iPSCs in clonal expansion.
5. Most laboratories use FBS-containing medium to derive fibro-
blasts. Chemically defined fibroblast medium is also commer-
cially available (ATCC® Primary Cell Solutions™ Media,
ATCC PCS-2-1-030; Fibroblast Growth Kit-Serum-free sup-
plement, ATCC PCS-201-040), or could be made in the labo-
ratory [4].
6. TrypLE™ contains recombinant protein, and can be neutral-
ized by simple dilution with basal medium. We recommend
TrypLE™ instead of Trypsin/EDTA for fibroblast dissocia-
tion to avoid using FBS or trypsin inhibitors.
7. EDTA is presently recommended to passage hESC and

iPSC. However, Dispase or TrypLE™ can also be used to pas-
sage cells.
8. STEMCCA lentivirus contains a polycistronic cassette that
overexpresses human OCT4, SOX2, KLF4, and c-MYC with
flanking LoxP sites. After reprogramming, the integrated cas-
sette can be excised with transient expressing Cre enzyme.
STEMCCA virus is commercially available through Millipore,
and it can also be produced in the laboratory. Other repro-
gramming methods can also be used in this protocol.
9. Gently mix the virus and avoid bubbles. Sendai virus can pro-
liferate in rodent cells, so avoid rodent cell culture or animals
in the area.
10. Matrigel® coating is not essential for reprogramming, and the
plate could be coated with fibronectin or recombinant
vitronectin.
11. Trypsin/EDTA can also be used in this step, and FBS-
containing medium is usually used to neutralize the enzyme
activity.
12. Most laboratories use 10 cm plates for reprogramming; how-
ever, we recommend using 6-well plates for the procedure.
13. Reprogramming medium 1 is designed to maintain optimal
fibroblast proliferation, which benefits reprogramming effi-
ciency. It should be used until the confluence reaches ~40%.
Many reprogramming cells are often visible 3 days after trans-
duction, but the majority of them will not mature to form true
iPSC colonies.
14. Reprogramming medium 2 is used for the maturation of
iPSCs. The medium is usually efficient enough for obtaining
iPSCs. However, some patient lines may be initially repro-
grammed well, but most of those cells die in a few days. We
often add sodium butyrate to boost the maturation of iPSC
colonies [4, 9].
15. It is important to control fibroblast confluence during repro-
gramming since over-crowding of cells on plates inhibits the
emergence of iPSC colonies. A plate can be passaged once, if
the plate is too confluent 1 week after transduction. Use 2
wells of cells for secondary passaging with TrypLE™, and plate
with 1× ROCK inhibitor.
16. Fibroblasts can secrete factors beneficial to iPSCs, so it is not
necessary to change the medium every day in the first 20 days
following reprogramming.
17. If starting fibroblasts proliferate quickly, and reprogramming
efficiency is very high, it can result in more than 100 colonies
in each well. The increased colony number will result in a
buildup of cellular waste products, altering medium pH and

turning the culture medium yellow every 24 h. In this situa-
tion, it is important to passage 1 or 2 wells of cells (1:3) with
EDTA. If cells are not passaged, it is important to change
medium every day.
18. It is essential to wait at least 20 days to allow the iPSCs to
mature. Seemingly, large iPSC colonies often appear 2 weeks
after transduction, but most of these early colonies cannot sur-
vive after mechanical isolation. Cells often survive much better
3 weeks after transduction. We usually pick colonies between
25 and 30 days.
19. It is important to cut the colonies under a microscope, and
plate the cells as small colonies. Big sheets of cells often float
in medium, and cannot settle efficiently on Matrigel®. Even
though picked colonies usually attach to the plate efficiently,
we often add ROCK inhibitor as insurance when plating.
20. We usually passage cells 2–3 days after picking with EDTA.
Acknowledgment
This work was supported by the NIH Center for Regenerative

Medicine and the National Heart, Lung and Blood Institute,
and G.C. is also supported by the University of Macau
(MYRG2015-00228-FHS). Jeanette Beers helped to establish
and edit the protocol.
References
1. Takahashi K, Tanabe K, Ohnuki M et al (2007) 6. Nishimura K, Sano M, Ohtaka M et al (2011)
Induction of pluripotent stem cells from adult Development of defective and persistent Sendai
human fibroblasts by defined factors. Cell virus vector: a unique gene delivery/expression
131:861–872 system ideal for cell reprogramming. J Biol
2. Takahashi K, Yamanaka S (2006) Induction of Chem 286:4760–4771
pluripotent stem cells from mouse embryonic 7. Beers J, Linask KL, Chen JA et al (2015) A
and adult fibroblast cultures by defined factors. cost-effective and efficient reprogramming plat-
Cell 126:663–676 form for large-scale production of integration-
3. Yu JY, Vodyanik MA, Smuga-Otto K et al free human induced pluripotent stem cells in
(2007) Induced pluripotent stem cell lines chemically defined culture. Sci Rep 5:11319
derived from human somatic cells. Science 8. Yu JY, Hu KJ, Smuga-Otto K et al (2009)
318:1917–1920 Human induced pluripotent stem cells free of
4. Chen G, Gulbranson DR, Hou Z et al (2011) vector and transgene sequences. Science
Chemically defined conditions for human iPSC 324:797–801
derivation and culture. Nat Methods 8:424–429 9. Mali P, Chou BK, Yen J et al (2010) Butyrate
5. Fusaki N, Ban H, Nishiyama A et al (2009) greatly enhances derivation of human induced
Efficient induction of transgene-free human pluripotent stem cells by promoting epigenetic
pluripotent stem cells using a vector based on remodeling and the expression of pluripotency-
Sendai virus, an RNA virus that does not inte- associated genes. Stem Cells 28:713–720
grate into the host genome. Proc Jpn Acad Ser
B Phys Biol Sci 85:348–362
Chapter 10
Culture, Adaptation, and Expansion of Pluripotent Stem

Cells
Jennifer L. Brehm and Tenneille E. Ludwig
Abstract
The ability of human pluripotent stem cells (hPSCs) to proliferate indefinitely in culture while maintaining
their pluripotent properties makes them a powerful tool for use in research, and provides tremendous
potential for diagnostic testing, and therapeutic application. Success in these areas, however, is dependent
on the ability to effectively expand them in long-term culture while preserving their distinct nature.
Contained in this chapter are detailed protocols for the feeder-independent culture and expansion of
hPSCs using mTeSR1 medium and Matrigel matrix, and guidelines for the successful transfer of those cells
to alternative platforms. These protocols have been used widely by laboratories around the world to suc-
cessfully expand hPSCs for long-term culture while maintaining their undifferentiated, pluripotent state.
Key words Pluripotent stem cell culture, Feeder-independent culture, Thawing, Passaging
1 Introduction
hPSCs have the potential to change the landscape of regenerative

medicine. They promise to be a tremendously powerful tool for
research into basic biological systems, pharmaceutical screening,
and drug development, and for understanding and treating dis-
ease. However, to fully exploit these capabilities, the cells must be
able to be reliably expanded in a way that preserves their unique
qualities. To maximize efficiency in the laboratory, the culture sys-
tem selected must be reasonably consistent, meaning that it must
be free of highly variable components that require intense screen-
ing such as serum and feeder layers common in many of the early
culture platforms for stem cell proliferation [1–3]. Feeder layers in
particular can be problematic, as in addition to their variability they
are also labor intensive to produce, and the routine production
required to support a standard research laboratory can consume
significant technical resources. Finally, ideally, the culture methods
chosen must be effective for a wide variety of lines generated using
139
140 Jennifer L. Brehm and Tenneille E. Ludwig
different derivation methods, and easily replicated across multiple

laboratories around the globe.
The mTeSR1/Matrigel culture platform presented here meets
all of these needs. Based on a culture system developed through
more than 4 years of careful optimization—including evaluations
of the effect of pH, atmosphere, osmolarity, culture substrate, and
more than 85 growth factors individually and in combination
[4]—mTeSR1 medium was designed with the basic research labo-
ratory in mind. As a more defined medium, it allows for further
optimization and investigation wherein the researcher can have
confidence that the effects observed are a result of the treatments
presented over time, and not due to variation within culture.
Because it can be used without feeder cells to culture hPSCs
directly or condition media for culture, scale-up capability is not
limited to the feeder production capacity of the laboratory. In
combination with the reasonably priced Matrigel matrix [5], it cre-
ates a culture platform that strikes a balance between consistency
and cost that makes it a reasonable selection for the majority of
hPSC research and preclinical work today. Evaluated using multi-
ple cell lines in a number of laboratories around the globe [6], this
feeder-independent system, while not completely defined due to
the inclusion of Matrigel, is robust, simple, efficient, and effective
at maintaining the undifferentiated, pluripotent state of the hPSC
following long-term culture.
Maintaining stability during expansion is critical. Previous
studies indicate that expansion techniques that result in cultures
being dissociated into single cells at passage may accumulate karyo-
typic abnormalities at a faster rate than cells passaged in clumps
[7–9]. While manual passaging results in the most consistent size
of clumps at passage, it is labor intensive, and not compatible with
the large-scale production required for potential industrial or ther-
apeutic applications. Alternative bulk passaging methods for
hPSCs, both enzymatic (e.g., collagenase, dispase) and non-
enzymatic (EDTA), can be used to passage large quantities of cells
with less risk of karyotypic instability. While the use of EDTA as
described in this protocol will result in small clumps of cells at pas-
sage, it demonstrates significant advantages over more common
alternative passaging reagents. Unlike collagenase and dispase,
which must be well rinsed from the cells to avoid cell membrane
damage, EDTA requires no washing and is gentler on the cultures,
resulting in greater recovery and viability. This allows for a signifi-
cant reduction in the time required to scale cultures, and an associ-
ated reduction in costs.
Cells cultured using the protocol as described here (mTeSR1
medium, Matrigel matrix, EDTA passaging reagent) have been
used to create clinical grade cell banks that maintain a normal
karyotype (by G-band), normal marker expression (>90% Oct3/4,
SSEA-3, SSEA-4, Tra-1-60 and Tra-1-81; <5% SSEA-1), and
Culture, Adaptation, and Expansion of Pluripotent Stem Cells 141
demonstrate pluripotency (by teratoma production and identification

of all three germ layers) following 40 passages of culture.
Additionally, comparative genome hybridization (CGH) per-
formed at the initiation of culture and again 40 passages later is
consistent with a normal karyotype and shows consistent changes
in relation to the reference standard with no aberrations greater
than 1 Mb in either sample, further indicating genetic stability over
long-term culture. We consistently use this protocol to scale up for
cell banking, and can expand from a single vial (~1 million cells) to
>1 × 109 cells in three to five passages depending on cell line.
While this protocol is written specifically for expansion in
mTeSR1 on Matrigel [6], it is important to note that a number of
other alternatives exist for both medium and matrix options for
feeder-independent expansion [6]. Specifically for matrix material,
while we recommend Growth Factor Reduced (GFR) Matrigel from
BD, we have also had success using this protocol in combination
with hESC-Qualified Matrix Matrigel (Corning, Corning, NY,
USA), Geltrex (Life Technologies, Carlsbad, CA, USA), and
Vitronectin (R&D Systems, Minneapolis, MN, USA). Likewise,
while we routinely use mTeSR1, we have had success using this pro-
tocol with MEF-conditioned medium [10], E8 medium [11], and
with minor modifications, StemPro Medium (Life Technologies).
The culture methods presented here are modified from those origi-
nally published for use with mTeSR medium [4, 5, 12].
2 Materials
2.1 Matrigel Aliquot 1. Growth Factor Reduced (GFR) Matrigel (Corning, Corning,
Components NY, USA: catalogue number 350234).
2. Ice bucket and ice.
3. Pipetman and tips.
4. 1.5 mL Eppendorf tubes.
2.2 Matrigel Plate 1. Matrigel aliquot (from Subheading 3.1).

Components 2. DMEM/F-12 medium.
(See Note 1)
3. 6-well plate (BD: catalogue number 08-772-1B).
4. 15 mL centrifuge tubes (Corning, Corning, NY, USA: cata-
logue number 430052).
5. 5.5 mL serological pipettes (Fisher Scientific, Waltham, MA,
USA: catalogue number 13-678-20D).
2.3 Thawing 1. Matrigel plate (from Subheading 3.2).

Materials 2. 15 mL centrifuge tubes (Corning, Corning, NY, USA: catalogue
number 430052).
3. 5.5 mL serological pipettes (Fisher Scientific, Waltham, MA,

USA: catalogue number 13-678-20D).
4. Forceps.
5. 37 °C water bath.
6. 95% ethanol.
7. Pasteur pipettes (Fisher, 13-678-20D), sterilized.
8. mTeSR1 medium (Stem Cell Technologies, Vancouver, BC,
Canada: catalogue number 05850) (see Note 2).
2.4 Culture 1. Matrigel plate (from Subheading 3.2).

and Expansion 2. mTeSR1 medium (Stem Cell Technologies, Vancouver, BC,
Materials Canada: catalogue number 05850) (see Note 2).
3. Microscope objective marker (Nikon, Melville, NY, USA: cata-
logue number MBW10020) (see Note 3).
4. Versene, 0.02% (Lonza, Walkersville, MD, USA: catalogue
number 17-711-E) (see Note 4).
5. Pasteur pipettes (Fisher: catalogue number 13-678-20D).
Sterilize prior to use.
6. 15 mL centrifuge tubes (Corning, Corning, NY, USA: cata-
logue number 430052).
7. 5.5 mL serological pipettes (Fisher, Pittsburgh, PA, USA: cata-
logue number 13-678-20D).
3 Methods
All reagent preparation and cell culture work should take place in a
Class 2 Biological Safety Cabinet (biosafety cabinet) unless other-
wise specified. All media and reagents should remain sterile. All
incubations and culturing should take place in a 37 °C incubator
with a humidified atmosphere of 5% CO2 in air (see Note 5).
3.1 Aliquot Matrigel 1. Thaw bottle of GFR Matrigel matrix overnight at 4–8 °C on
for Use ice according to the manufacturer’s recommendations.
2. At least 1 h prior to aliquoting GFR Matrigel, place pipette tips
and prelabeled 1.5 mL tubes in freezer (−20 °C) to chill.
Assure that DMEM/F-12 medium is chilled to 4–8 °C.
3. Keeping Matrigel on ice, use chilled tips to aliquot 0.5 mg
GFR Matrigel into 1.5 mL tubes on ice (see Note 6).
4. Immediately freeze tubes at −80 °C. Aliquots can be stored for
a minimum of 1 year prior to use (see Note 7).
3.2 Prepare When plating Matrigel, it is critical to work quickly. Allowing the
Matrigel Plate Matrigel to warm before it is plated will result in clumps, uneven
coating, and reduced attachment.
1. Place a sterile 6-well plate, sterile 15 mL conical tube, and sterile

ice-cold DMEM/F-12 medium into the biosafety cabinet.
2. Add 11 mL of ice-cold DMEM/F-12 medium to the conical
tube.
3. Remove Matrigel aliquot from freezer, and working in the
biosafety cabinet, immediately add 1 mL of ice-cold DMEM/F-12
to the aliquot.
4. Gently pipet up and down to thaw the Matrigel. This is best
accomplished using a pipetman with a 1 mL tip. Immediately
transfer Matrigel solution to the conical tube containing 11 mL
ice-cold DMEM/F-12 (total volume should now be 12 mL).
5. Mix by gently pipetting up and down using a 5 mL pipette,
and immediately plate 2 mL of solution into each well of the
6-well plate (see Note 8).
6. Plates may be used on the same day, or prepared up to 2 weeks
in advance. Allow plates to rest for 1 h at room temperature
prior to use or storage. Plates may be stored in a 37 °C incubator
or wrapped in parafilm and stored at 4–8 °C. Plates stored in
the incubator may be used immediately, while plates stored at
4–8 °C should be warmed for at least 1 h to room temperature
prior to use.
3.3 Thaw It is generally recommended that cells be thawed into the same
and Culture culture system that was used immediately prior to freeze, and well
Pluripotent Stem Cells established prior to transfer to an alternate culture system (see Note 9).
This portion of the protocol assumes that the culture was frozen
using the mTeSR1/Matrigel system.
1. Determine surface area into which the material should be
thawed. Using the correct plating density is critical to achiev-
ing a successful thaw. Generally, one vial will be thawed into a
single well of a 6-well plate; however, different providers may
have different recommendations to achieve the best results
(see Note 10).
2. Aspirate excess Matrigel plating solution from fresh or stored
Matrigel plate and add 2 mL fresh room temperature mTeSR1
medium to each well to be used. Label plate appropriately, and
place in an incubator.
3. Remove vial from LN2 storage, and using long forceps immerse
it into a 37 °C water bath without submerging the cap (see
Note 11).
4. Swirl the vial gently, being careful not to wet the cap. When
only a small ice crystal remains remove the vial from the water
bath and (check to assure that the cap is tight) immerse the
vial into a 95% ethanol bath to clean the outside of the tube
(see Note 12).
5. Place tube immediately into the biosafety cabinet, and allow to

air dry for 15–30 s.
6. Using a pipet, gently transfer the cells into a 15 mL conical
tube being careful not to disrupt the cell clumps more than
necessary.
7. While gently swirling the cells, slowly add 11 mL of mTeSR1
dropwise to the cell suspension (see Note 13).
8. Centrifuge the cells at 200 × g for 5 min.
9. In the biosafety cabinet, using a sterile pipette, aspirate and
discard the supernatant, and resuspend the cell pellet in 0.5 mL
mTeSR1 for every well that will be plated. Example: Provider
recommends thawing one vial into 4 wells,
4 × 0.5 mL = 2 mL. Resuspend pellet into 2 mL mTeSR1.
10. Place 0.5 mL of cell suspension into each well of the prepared
culture plate. Immediately return to incubator, moving plate
front to back and side to side to evenly distribute cells through-
out the well. Avoid circular movements to prevent pooling in
center of well.
11. The following day, examine and feed the plate 2 mL of medium
per well (remove spent medium and replace with fresh mTeSR1).
Feed daily until ready to passage or freeze (see Note 14).
3.4 Passage Determining appropriate passage timing in feeder-independent cul-

and Expand ture systems, such as mTeSR1, is critical for optimal culture. Passaging
Pluripotent Stem Cells too early results in reduced attachment, while allowing the culture to
overgrow leads to increased differentiation and reduced attachment.
Passage cells when the center of the colonies begins to become dense,
appearing brighter than the edge when viewed under a phase contrast
microscope. When initially using this culture system, it may be help-
ful to passage sister wells of a single culture on successive days and
observe them for several days following passage to best determine the
most appropriate passage timing.
1. View culture to be passaged or expanded under the microscope,
and use the microscope objective marker to indicate areas of
differentiation to be removed prior to passage (see Note 15).
2. Determine passaging ratio. In general, passing ratios in the
mTeSR1/Matrigel culture system will average 1:15 (one well
can be split into 15 wells at passage). Less dense cultures
should be split at a lower ratio, perhaps 1:10, and more dense
cultures can be split at a ratio of 1:20 or higher. Use the passage
ratio to determine the number of plates (or other culture
vessels) to prepare (see Note 16).
3. Prepare plates by aspirating the excess Matrigel plating solu-
tion from fresh or stored Matrigel plates and add 2 mL fresh
mTeSR1 to each well to be used. Label the plate appropriately
and place in the incubator.
4. Aspirate the spent medium from each well of the culture to be

passaged with a sterile Pasteur pipette. At least one well of the
culture should not be passaged, but saved to use as a “back-up
well.” This will mitigate the risk associated with passaging, and
allow the culture to be saved if cells do not attach properly or
are contaminated following passage.
5. Rinse each well to be passaged with 1 mL room temperature
Versene. Aspirate Versene using a sterile Pasteur pipette (see
Note 17).
6. Add 1 mL Versene to each well to be passaged, and incubate at
room temperature for 5–7 min.
7. While incubating, determine the appropriate volume of cell
suspension to add to each new well to achieve the desired split
ratio. This is accomplished by dividing the anticipated collec-
tion volume (assume 2–3 mL/well to be collected, plus
another 2–3 mL for rinsing) by the total number of wells that
could be produced using the following formula:
(Anticipated collection volume)/(# wells being passaged)
(# wells per well based on split ratio)
Example: If 10 mL mTeSR1 is used to collect 3 wells being
split at a 1:15 ratio, the formula is: 10 mL/(3)
(15) = 10 mL/45 = 222 μL
Therefore, in this example, 222 μL of cell suspension would go
into each freshly prepared well.
8. Following incubations, aspirate Versene from adherent cells
using a sterile Pasteur pipette, touching the tip of the pipette
to the plate to remove any marked areas of differentiation (see
Note 18).
9. To remove cells from plate, use a 5 mL pipette with 1–3 mL of
medium to gently wash the cells from each well. Cells should
wash off easily without scraping, and care should be taken not
to further disrupt the cell clumps (see Note 19). Rinse plate to
collect residual cells. Pool the cell suspension into a sterile con-
ical tube.
10. Add the appropriate volume of cell suspension to each well of
the freshly prepared plate (as calculated in Subheading 3.4,
step 7).
11. Return plate to incubator moving plate side to side and front
to back to evenly distribute cells within each well, avoid circu-
lar movements to prevent pooling in center of well. Incubate
overnight undisturbed (see Note 20).
12. The following day, replace spent medium with 2 mL fresh culture
medium. Feed daily until ready to passage or freeze.
Table 1
Recommendations for transferring hPSC cultures from mTeSR1 on Matrigel to an alternate culture
platform
Timing of
New culture platform transfer Tips for transitioning
MEF-Based Culture At passage Harvest cells using dispase [6]. Resuspend cells at harvest in hES
System Medium (DMEM/F-12 with KOSR). Increase density at
plating (e.g., if split ratio was 1:10, split at 1:5), and plate onto
a fresh MEF feeder layer. Continue culture as appropriate for
MEF-based culture
Conditioned At passage Resuspend cells at harvest in conditioned medium. Plate as usual
Medium/Matrigel onto Matrigel. Continue culture as appropriate for the specific
conditioned medium being used
E8 Medium/Matrigel At passage Resuspend cells at harvest in E8 Medium, ROCK inhibitor
optional. Increase density at initial plating (e.g., if the split
ratio was 1:10, split at 1:8), and plate directly onto Matrigel.
Continue culture per manufacturer’s instructions
E8 Medium/ At passage Resuspend cells at harvest in E8 Medium plus 10 μM Rock
Vitronectin Inhibitor. Increase density at initial plating (e.g., if the split
ratio was 1:10, split at 1:6), and plate directly onto
Vitronectin. Continue culture per manufacturer’s instructions
StemPro/GelTrex At passage Resuspend cells at harvest in StemPro Medium. Increase density
at initial plating (e.g., if the split ratio was 1:10, split at 1:8),
and plate directly onto Geltrex. Continue culture per
manufacturer’s instructions
3.5 Transferring Transferring between culture platforms is occasionally advantageous,

Between Culture and can be easily achieved. In general, transitions can be made at
Platforms passage with no adaptation time by making minor adjustments in
plating density.
1. To transfer from alternate culture platforms to the
mTeSR1/Matrigel, passage the cultures a bit more densely
than a standard passage (e.g., if split ratio was 1:4, plan to pas-
sage at 1:3 for the initial transfer). Resuspend cells at harvest in
mTeSR1, and plate directly onto Matrigel. Refresh spent
medium daily with 2 mL fresh mTeSR1 until ready to passage
or freeze (see Note 21).
2. To transfer from mTeSR1/Matrigel to alternate feeder-
independent platforms, harvest material as indicated previously
(Subheading 3.4, steps 4–6), and consult Table 1 when consider-
ing plating volumes (step 7). Manufacturer’s recommendations
should be followed when transferring to an alternate platform not
listed below, and primary cultures should be continued in parallel
until the new platform is well established in all cases.
4 Notes
1. Multiple matrix options exist for feeder-independent expansion

of human pluripotent stem cells. While this protocol is written
specifically for expansion on GFR Matrigel, we have also had
success using other Matrigel formulations (Corning) as well as
Geltrex (Life Technologies) at the same concentration, and
Vitronectin (R&D Systems) plated at 2–5 μg/well [13].
2. While this protocol has been written specifically for expansion
in mTeSR1, it is important to note that a number of alterna-
tives exist for long-term, undifferentiated proliferation of plu-
ripotent stem cells. We have successfully used this protocol in
combination with MEF-conditioned medium [10], StemPro
(Life Technologies) and E8 medium [11].
3. While not required, this marker aids significantly in identifying
small areas of differentiation. It allows a small circle to be inked
onto the bottom of the plate, clearly identifying areas of
differentiation that can then be easily removed in the sterile
environment of the biosafety cabinet prior to passaging cells.
Adaptors are available to allow attachment to non-Nikon
microscopes.
4. Commercial products are listed for convenience. To make an
equivalent EDTA splitting medium in house, add 500 mg
EDTA (Sigma, Saint Louis, MO, USA: catalogue number
E6758) to 250 mL PBS−/− and stir to dissolve (this may take
several hours, and may require warming to dissolve completely).
pH solution to 7.2, adjust osmolarity to 340 mOsMol, and
filter sterilize. Solution may be stored at room temperature for
up to 6 months.
5. Some groups, including ours, have had increased success using
low oxygen (≤5% O2: [4]). While we do see increased attach-
ment in low oxygen environments, to achieve any additional
advantage, the atmosphere must be well controlled and remain
stable. If the atmosphere is allowed to fluctuate with repeated
opening and closing of the doors, etc., the advantages diminish
rapidly, and cultures may be negatively impacted.
6. Be aware that the protein concentration in Matrigel varies
among lots; therefore, the exact volume of Matrigel necessary
to obtain 0.5 mg will vary with the lot used. If more than one
plate is routinely prepared at a time, larger aliquots may be
prepared.
7. We have tested Matrigel following more than 3 years in storage
at −80 °C with no effective reduction in performance.
8. If plates will be used the same day that they are prepared,
0.5 mg Matrigel may be diluted in a total of 6 mL DMEM/F-12
medium and plated at 1 mL/well into 6-well plates. The addi-
tional volume is required to prevent drying of the wells on
storage.
9. Cells will be adapted to the culture system in which they are
frozen. To achieve the best results at thaw, reducing stress to
the cells is critical. Using the same culture platform for thaw-
ing that was used at the time of freeze will reduce stress on the
culture at thaw, and result in better initial attachment and pro-
liferation. Cultures should be well established in the initial cul-
ture system before transferring to any new format. Following
transfer, it is advisable to maintain the initial culture in the
original conditions in parallel until it is clear that the transition
was successful.
10. If provider recommends a culture vessel other than a 6-well
plate, use 0.009 mg Matrigel per cm2 of surface area to be
coated. Standard alternate culture vessel configurations are
included in Table 2.
11. Be sure to wear appropriate personal protective equipment
including safety glasses, as vials can sometimes explode. Take
care not to wet the cap, as this will increase the potential for
contamination of the material as you remove it from the vial.
12. Many labels are not ethanol resistant. Be sure to record all
pertinent information from the vial label prior to immersing
into ethanol.
13. Adding the medium slowly and a little at a time is important in
reducing osmotic shock to the cells as they thaw. It should take
several minutes to add all 11 mL of medium to the cell pellet.
Failure to control osmotic shock may increase death loss and
reduce attachment post thaw.
Table 2
Recommendations for coating common alternative culture vessels with
Matrigel matrix
Surface area Concentration DMEM/F-12

Culture vessel of vessel (cm2) of Matrigel (mg) Diluant (mL)
6-well plate 9.6 (1 well) 0.08 (for 1 well) 2
35 mm dish 9.6 0.08 2
10 cm dish 78 0.68 5
T75 flask 75 0.65 6
T25 flask 25 0.22 4
14. For daily feeding, when using mTeSR1, warming medium prior
to feeding is not necessary, and cells may be fed with medium
straight out of the refrigerator at 4–8 °C. Side-by-side testing
over ten passages shows no significant effect when compared to
feeding using warm medium. Additionally, 1 day per week of
overfeeding is acceptable. When overfeeding, replace spent
medium with 4 mL of fresh mTeSR1, and do not feed the
following day. Overfeeding is not recommended immediately
preceding passaging, but can be done at any other time.
15. If percentage of differentiation in the plate exceeds 10–20%,
alternate methods of removal are advised.
If >20% but <50% of the plate is differentiated, then areas of
differentiation should be removed from the culture with a modi-
fied pipette or micropipette tip. Scrape differentiated areas
from the plate, and remove from the culture by rinsing the plate
and adding fresh DMEM/F-12 medium prior to passaging.
If >50% of the culture is differentiated, then the undifferen-
tiated portions of the colony should be manually removed using
a modified pipette, a needle, or a SweMed cutting tool (Vitrolife,
Göteborg, Sweden: catalogue number 14602). Carefully cut
mature, undifferentiated portions of colonies into small
(~30–50 cell) clumps, nudge off the plate carefully, and transfer
to freshly prepared plate for continuing culture.
Be aware when determining whether to use these tech-
niques to remove differentiation from a continuing culture
that these actions are, by their very nature, selection events.
Increasing selection events in a pluripotent cell culture can
drive a cell line toward an abnormal karyotype. These more
extreme techniques for ridding a culture of differentiation,
therefore, should be procedures used when absolutely necessary
to maintain an irreplaceable or otherwise valuable culture, and
not performed as routine practice. When significant selection is
required, G-band analysis to confirm normal karyotype of the
culture is recommended.
16. When using E8 and Vitronectin, reduce split ratio slightly to
an average of 1:8.
17. Rinsing is critical to remove any residual calcium from culture
medium and allow the proper functioning of the Versene.
Room temperature Ca and Mg free PBS may also be used to
rinse plates if Versene is limited.
18. If cells have become free floating, collect in a conical tube and
centrifuge at 200 × g for 5 min. Resuspend in mTeSR1
medium as calculated in Subheading 3.4, step 7, and distribute
into plates.
19. It is particularly important not to dissociate the cells into single
cells at passage. Karyotypically normal hPSCs do not survive
well when individualized. Certain karyotypic abnormalities
confer an advantage in cloning efficiency. This means that if the

culture is passaged as a single-cell suspension, karyotypically
abnormal cells within the culture may have a selection advan-
tage, and could rapidly overtake the culture. This selection
advantage may account for the number of reoccurring karyo-
typic abnormalities common to hPSCs [14]. When passaged as
clumps, this advantage is greatly reduced.
20. Avoid opening and closing the incubator doors for the first sev-
eral hours after passaging cells, as the associated movement of the
incubator will cause the cells to pool in the center of the wells.
21. If transferring to a feeder-independent culture system from an
MEF-based system, be aware that it is normal for MEFs to
persist in the culture for up to two full passages following
transfer.
Acknowledgment
This work was supported by funding from the Wisconsin Alumni

Research Foundation.
References
1. Thomson JA, Itskovitz-Eldor J, Shapiro SS et al 8. Buzzard JJ, Gough NM, Crook JM, Colman A
(1998) Embryonic stem cell lines derived from (2004) Karyotype of human ES cells during
human blastocysts. Science 282:1145–1147 extended culture. Nat Biotechnol 22:381–382
2. Reubinoff BE, Pera MF, Fong CY et al (2000) 9. Mitalipova MM, Rao RR, Hoyer DM et al
Embryonic stem cell lines from human blasto- (2004) Preserving the genetic integrity of
cysts: somatic differentiation in vitro. Nat human embryonic stem cells. Nat Biotechnol
Biotechnol 18:399–404 23:19–20
3. Amit M, Carpenter MK, Inokuma MS et al 10. Xu C, Inokuma MS, Denham J et al (2001)
(2000) Clonally derived human embryonic Feeder-free growth of undifferentiated human
stem cell lines maintain pluripotency and pro- embryonic stem cells. Nat Biotechnol 19:
liferative potential for prolonged periods of 971–974
culture. Dev Biol 227:271–278 11. Chen G, Gulbranson DR, Hou Z et al (2011)
4. Ludwig TE, Levenstein ME, Jones JM et al Chemically defined conditions for human iPSC
(2006) Derivation of human embryonic stem derivation and culture. Nat Methods 8(5):
cells in defined conditions. Nat Biotechnol 424–429
24(2):185–187 12. Ludwig TE, Thomson JA (2007) Defined,
5. Ludwig TE, Bergendahl V, Levenstein ME feeder-independent medium for human embry-
et al (2006) Feeder-independent culture of onic stem cell culture. Curr Protoc Stem Cell
human embryonic stem cells. Nat Methods Biol:1C.2.1–1C.2.16
3(8):637–646 13. Braam SR, Zeinstra L, Litjens S et al (2008)
6. International Stem Cell Initiative Consortium Recombinant vitronectin is a functionally
(2010) Comparison of defined culture systems defined substrate that supports human embry-
for feeder free propagation of human embryonic stem cell self-renewal via alphavbeta5 inte-
onic stem cells. In Vitro Cell Dev Biol Anim grin. Stem Cells 26(9):2257–2265
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Chapter 11
Cryobanking Pluripotent Stem Cells

Jeremy M. Crook, Eva Tomaskovic-Crook, and Tenneille E. Ludwig
Abstract
Cryobanking human pluripotent stem cells (hPSCs), be they human embryonic (hESCs) or induced
pluripotent stem cells (iPSCs), is essential for their use in research and cell-based therapeutics. Working
and master cell banks can be generated with a desired level of quality assurance applied during cell freezing
and storage. Conventional vitrification has evolved to more advanced control rate freezing, culminating in
a myriad of published protocols with variable proficiencies and clinical efficacies. Notwithstanding,
standardized and reliable protocols are necessary for basic science through to applied research and clinical
product development. This chapter details several methods for hPSC cryopreservation, suitable for routine
application, high-quality research, and adaptable for clinical compliance.
Key words Human pluripotent stem cells, Vitrification, Control rate freezing, Cryopreservation, Banking
1 Introduction
The ability to maintain frozen stocks of hESCs and iPSCs is a critical

component to their use in research and translation. Early protocols
for cryopreservation were based on methods used by fertility clin-
ics for freezing embryos and have evolved for improved research
and clinical compliance [1, 2]. Therefore, emphasis has been placed
on increased cell viability with thawing after freezing, scalability,
and the use of defined components that are amenable to regulatory
approval for therapeutics. Importantly, the unique in vitro growth
characteristics of hPSCs have required specialized protocols for
their processing for freezing and banking.
While the multitude of approaches reflects the need to develop
better, efficient, and safer methods of freezing and banking, it is
important to be able to discern suitable and reliable protocols that
can be easily employed across different laboratories as standardized
procedures for consistent and reproducible outcomes. Ideally,
options should include protocols that satisfy the requirements of
basic research and the more rigorous requirements of applied
research. Here, we describe a selection of methods that are reliable
and adaptable for clinical product development [3].
151
2 Materials
2.1 Reagents 1. Dulbecco’s Modified Eagle Medium (DMEM; Gibco, Life

Technologies, Cat. no. 11960-044).
2.1.1 Reagents
for Vitrification Method 2. hPSC Qualified Foetal Bovine Serum (FBS; Gibco, Life
Technologies, Cat. no. 10439-024).
3. HEPES buffer, 1 M (Gibco, Life Technologies, Cat. no.
15630-106).
4. Sucrose (Sigma-Aldrich, Cat. no. S7903).
5. Ethylene glycol (Sigma-Aldrich, Cat. no. 324558).
6. Dimethyl sulfoxide (DMSO; Sigma-Aldrich, Cat. no. D1435).
7. LN2.
2.1.2 Reagents for Slow 1. Defined FBS (Hyclone, Cat. no. SH30070.01, or
Freezing Method equivalent).
2. DMSO, 10 mL ampoules (Sigma-Aldrich, Cat. no. D2438).
3. Cell culture medium (see Note 1).
4. Harvest reagent (see Note 1).
5. DMEM/F-12 (Gibco, Life Technologies, Cat. no.
11330-057).
6. Isopropyl alcohol (Chem Supply, Cat. no. 323-4).
7. 95% Ethanol.
2.1.3 Reagents 1. Dulbecco’s phosphate buffered saline (DPBS, with Ca2+ and
for Controlled Rate Mg2+; Gibco, Life Technologies, Cat. no. 14040-133).
Freezing Method 2. Collagenase, Type IV (Gibco, Life Technologies, Cat. no.
17104-019).
3. hPSC culture medium.
4. CryoStor™ CS10 (Biolife solutions, STEMCELL Technologies,
Cat. no. 99-610-DV).
5. 95% Ethanol.
2.2 Labware 1. Vitrification straws (sterile; LEC instruments).

2.2.1 Labware 2. Syringe filter, 0.22 μm membrane (Millipore, Cat. no.
for Vitrification Method SLGP033RS).
3. 50 mL centrifuge tubes (Falcon, Cat. no. 35-2070).
4. 15 mL centrifuge tubes (Falcon, Cat. no. 35-2096).
5. 4-well cell culture plates (Nunc, Thermo Fisher Scientific,
Cat. no. 176740).
6. Glass capillaries (1.0 mm outer diameter; Harvard Apparatus,
Cat. no. GC100T-15; see Note 2).
Cryobanking Pluripotent Stem Cells 153
7. 4.5 mL Cryovials (Nunc, Thermo Fisher Scientific, Cat. no.

379146).
8. 18-Gauge needle (BD, Cat. no. 305196).
2.2.2 Labware for Slow 1. 5 mL sterile serological pipettes (Fisher, Cat. no. 13-678-27E).
Freeze Method 2. 10 mL sterile serological pipettes (Fisher, Cat. no. 13-678-27F).
3. 15 mL centrifuge tubes (Corning, Sigma-Aldrich, Cat. no.
430052).
4. Baked pasteur pipettes (Fisher, Cat. no. 13-678-20D).
5. 150 mL sterile filter unit (0.22 μm membrane) (Nalgene, Cat.
no. 565-0020).
6. 50 mL centrifuge tubes (Corning, Cat. no. 43029).
7. 150 mL receiver flask (Nalgene, Cat. no. 455-0150).
8. 1.5 mL Cryovials (Nunc, Thermo Fisher Scientific, Cat. no.
5000-1020).
9. 6-well cell culture plate (Nunc, Thermo Fisher Scientific, Cat.
no. 1483211).
10. 1 mL sterile serological pipettes (Fisher, Cat. no. 13-678-27B).
2.2.3 Labware 1. 15 mL centrifuge tubes (Falcon, Cat. no. 35-2096).

for Controlled Rate Freeze 2. 5 mL sterile serological pipettes (Fisher, Cat. no. 13-678-27E).
Method
3. 0.5 mL CBS High Security Straws (sterile; Cryo Bio System,
Cat. no. 014651).
4. Cell Scrapers (Corning, Falcon, Cat. no. 35-3086).
5. Prepared thawing plate or culture vessel.
6. Sterile scalpel or lab scissors.
2.3 Special 1. Light microscope.

Equipment 2. LN2 dewar.
2.3.1 Special Equipment 3. Bunsen burner.
for Vitrification Method 4. Aluminum cryocanes.
5. 18-Guage needles (BD, Cat. no. 305196).
6. 20 μL Pipettor (P20).
8. Long-term LN2 storage unit.
9. CO2 incubator.
10. Sterile biosafety cabinet.
2.3.2 Special Equipment 1. Sterile biosafety cabinet.

for Slow Freeze Method 2. −80 °C Ultra low freezer.
3. Centrifuge.
4. CO2 incubator.
5. Pipet-Aid (motorized pipette).
6. Ice bucket.
7. LN2 storage tank.
8. Isopropanol freezing units (Nalgene, Thermo Fisher, Cat. no.
5100-0001 Cryo 1 °C “Mr. Frosty”) or equivalent non-isopropanol
freezing units (CoolCell: Corning, Cat. no. 432001).
9. Cryovial racks.
11. Safety glasses.
12. Metal forceps.
2.3.3 Special Equipment 1. Planar biological freezer (Planar Kryo 360-1.7).

for Controlled Rate 2. Pipet-Aid (motorized pipette).
Freezing Method
3. Ice bucket filled with ice.
5. Long-term LN2 Storage Unit.
7. Centrifuge.
8. SYMS Manual Sealing Unit for CBS Straws (Cryo Bio System,
Cat. no. 007213).
9. Forceps.
10. LN2 dewar.
11. Sterile scalpel or scissors.
12. Aspiration System.
13. CO2 incubator.
3 Methods
3.1 Vitrification Vitrification of hPSCs is derived from methods developed for

Method freezing blastocysts [1, 2]. Although associated with low throughput
and poor post-thaw cell viability (i.e., typically yields 1–10%
survival) it is widely used since it is easy to perform. In addition,
early reports employed open straw containment with the risk of
exposing cells to contaminating microbial and viral pathogens
before, during, and after LN2 [4]. However, more contemporary
and efficacious closed straw freezing can be employed, consistent
with current Good Laboratory Practice (cGLP) and current Good
Manufacturing Practice (cGMP) application toward clinical
compliance [3, 5]. All work should be performed in a sterile envi-
ronment (e.g., sterile biosafety cabinet).
3.1.1 Cryopreservation 1. Combine DMEM (15.6 mL), FBS (4 mL), and 1 M HEPES
Solutions (0.4 mL).
Base Solution 2. Filter solution through a syringe filter into a 50 mL tube.
3. Store up to 1 week at 4 °C.
1 M Sucrose Solution 1. Add 3.42 g sucrose to 6 mL Base Solution in a 15 mL tube.

2. Dissolve sucrose at 37 °C.
3. Bring volume to 8 mL with additional Base Solution.
4. Add 2 mL FBS.
5. Filter solution through a syringe filter into a fresh 15 mL tube.
0.2 M Sucrose Solution 1. Combine Base Solution (4 mL) and 1 M Sucrose Solution
(1 mL).
0.1 M Sucrose Solution 1. Combine Base Solution (4.5 mL) and 1 M Sucrose Solution
(0.5 mL).
10% Vitrification Solution 1. Combine Base Solution (2 mL) with ethylene glycol (0.25 mL)
and DMSO (0.25 mL; see Note 3).
20% Vitrification Solution 1. Combine Base Solution (0.75 mL) with 1 M Sucrose Solution
(0.75 mL), ethylene glycol (0.5 mL), and DMSO (0.5 mL;
see Note 3).
3.1.2 Vitrification 1. Prepare a 4-well plate (“Vitrification Plate”) with Base

Solution in well 1, 10% Vitrification Solution in well 2, and 20%
Vitrification Solution in well 3, at 37 °C.
2. Prepare 4.5 mL cryovials for step 14 below by punching a hole
through the side with an 18-gauge needle heated in a Bunsen
burner flame.
3. Prepare a rinsing plate with fresh preferred hPSC culture media
equilibrated to 37 °C and 5% CO2.
4. Transfer the cryovials to cryocanes and immerse in LN2.
5. All subsequent steps must be completed within 3 min for each
straw to be frozen.
6. Harvest hPSCs for freezing by microdissection, for subsequent
transfer of colony fragments to the rinsing plate (see Note 2).
7. Transfer the colony fragments using a P20 pipettor to well 1 of
the Vitrification Plate.
8. Prepare a fresh 20 μL drop of 20% Vitrification Solution on the

underside of the Vitrification Plate lid.
9. Transfer six to nine colony fragments from well 1 to well 2 of
the Vitrification Plate and leave for 1 min.
10. Transfer the colony fragments from well 2 to well 3 of the
Vitrification Plate and leave for 25 s.
11. Transfer the colony fragments in the smallest possible volume
to the 20 μL drop of 20% Vitrification Solution.
12. Using a P10 pipettor, remove the fragments in a 3 μL volume
from the 20 μL drop and expel as a separate small, peaked
droplet on the lid.
13. Apply the narrow end of a straw to the droplet at about a 30°
angle to the plane of the dish to draw the droplet into the straw
by capillary action. Do not attempt to recover any fragments
that are not drawn up.
14. Immediately plunge the straw into LN2, followed by transfer
to a designated cryovial in the LN2 (see Note 4).
15. Store in long-term LN2 unit until further required.
3.1.3 Thawing 1. Pre-warm a 4-well plate (“Thawing Plate”) to 37 °C with

Vitrified hPSCs 0.2 M Sucrose Solution in well 1, 0.1 M Sucrose Solution in
well 2, and Base Solution in wells 3 and 4.
2. Quickly transfer cryovial(s) containing frozen straw(s) from
LN2 storage canisters to a holding container filled with LN2.
Avoid thawing of frozen cells.
3. Remove a straw using forceps from the cryovial, again avoiding
thawing of the cells.
4. Working quickly, hold the straw between thumb and middle
finger at a slight angle, submerge the narrow end to well 1 of
the Thawing Plate.
5. Immediately following melting of the cell/media column,
place a finger on the top of the straw.
6. The cells/media column will be forced from the straw with gas
expansion. Expel any remnant liquid from the straw by placing the
tip of a P200 pipettor in the top end of the straw and ejecting.
7. After 1 min, transfer the colony fragments to well 2.
8. After 5 min transfer the colony fragments to well 3.
9. After 5 min transfer the colony fragments to well 4.
10. After 5 min, plate the fragments to prepared culture vessels for
recovery and expansion.
3.2 Slow The Slow Freezing method presented here is appropriate for banking
Freezing Method cells within the research lab and for distribution. Cells are cryopre-
served in 1.5 mL cryovials, which are readily storable in most
long-term LN2 storage units. In our hands, lots of up to 150 vials

are readily prepared regularly, but it is also very efficient for small-
scale (1–2 vials) freezes of contingency materials for use within the
lab. The protocol presented here is written for use with cell cultures
maintained in 6-well plates, but can also be used for alternate for-
mats by adjusting the media amounts based on the surface area of
the culture vessel. By making small adjustments as indicated, this
protocol can be used with TeSR, E8, and KOSR-based cell cultures
with equal success. All work should be performed in a sterile envi-
ronment (e.g., sterile biosafety cabinet).
3.2.1 Harvesting 1. For feeder-independent TeSR and E8-based cell cultures,

Reagent Versene (EDTA) is recommended.
2. For feeder-free KOSR-based cultures on Matrigel, Dispase
Solution is recommended. Dispase Solution is 36 mg Dispase
powder dissolved in 10 mL DMEM/F-12. Filter sterilize. This
will be enough solution to harvest ten confluent wells for
cryopreservation.
3. For KOSR-based cultures on murine fibroblasts (MEFs),
Collagenase Solution is recommended. Collagenase Solution is
10 mg Collagenase Type IV powder dissolved in 10 mL
DMEM/F12. Filter sterilize. This will be enough solution to
harvest ten confluent wells for cryopreservation.
3.2.2 Cryopreservation 1. For TeSR-based cultures, TeSR Cryopreservation Medium

Solution consists of 60% sterile TeSR-based medium, and 20% sterile
DMSO (see Note 3).
2. For E8-based cell cultures, E8 Cryopreservation Medium con-
sists of 80% sterile E8-based medium and 20% sterile DMSO
(see Note 3).
3. For KOSR-based cell cultures, KOSR Cryopreservation
Medium consists of 60% sterile FBS, 20% sterile complete
KOSR-based medium (such as hPSC medium), and 20% sterile
DMSO (see Note 3).
3.2.3 Slow Freezing 1. Determine number of vials to be cryopreserved (for TeSR and
E8-based cultures, assume 1.5 vials per well of confluent cells,
for KOSR-based cultures, assume a vial for each well of conflu-
ent cells), and label vials appropriately, including cell line name,
passage number, and date frozen at a minimum (see Note 4).
2. View all cells to be cryopreserved under the microscope, mark
and remove any differentiation as appropriate.
3. Remove spent culture medium from culture vessel.
4. Add 1 mL of appropriate Harvest Reagent to each well of a
6-well plate (adjusting as appropriate for alternate culture ves-
sels) to harvest cells.
(a) If using Versene, aspirate the Versene and add an additional

mL of Versene, and incubate the cells for 7–10 min at
room temperature.
(b) If using Dispase Solution, incubate cells for 7 min at 37 °C.
(c) If using Collagenase Solution, incubate cells for 5 min at
37 °C.
5. If Versene was used to incubate the cells (otherwise, if Dispase
or Collagenase was used, skip to step 12 below), following
incubation, carefully aspirate Versene from each well, harvest-
ing one plate at a time. The cells should remain adhered to the
bottom of the well.
6. Working quickly to prevent wells from drying out, using a
5 mL pipette, add 3 mL preferred hESC culture medium
(TeSR or E8-based medium) to the first well of the plate, and
rinse the cells off by quickly pipetting the medium across the
cells in a back and forth pattern.
7. Use the same 3 mL medium to rinse each additional well in the
plate, being careful to pipet as little as possible to prevent
breaking up the colonies. Transfer the cell-containing medium
to a 50 mL conical tube and proceed with harvesting all addi-
tional plates, pooling cells from subsequent plates in the same
50 mL conical tube.
8. Rinse each plate with 1.5 mL preferred culture medium, and add
the resulting suspension to the pooled cells in the same 50 mL
conical tube. DO NOT CENTRIFUGE. Skip to step 26.
9. If Dispase Solution was used to incubate the cells (otherwise,
if Collagenase Solution was used, skip to step 19 below), care-
fully aspirate Dispase Solution from the plate; the cells should
remain adhered to the plate (see Note 5).
10. Rinse the adherent cells by adding 1 mL DMEM/F12 medium
to each well and aspirating to remove. Repeat this rinse two
additional times for a total of three rinses.
11. Add 1 mL preferred culture medium to each well. Using a
5 mL pipette, take up the medium from multiple wells, and use
it to harvest the colonies by slowly pipetting the medium while
scraping the plate with the end of the pipette. Be careful not to
break up the colonies—cells will recover from thaw more
efficiently if larger aggregates can be maintained.
12. Pool cells from each plate into a 50 mL conical tube, repeating
for each plate and adding to the same 50 mL conical tube.
13. Wash each plate with an additional 3 mL of preferred culture
medium, and add suspension to the pooled cells in the 50 mL
conical tube.
14. Centrifuge pooled cells at 200 × g, redistributing into smaller
pools if necessary.
15. Aspirate supernatant, and resuspend cell pellet in preferred

hPSC medium equal to 3 mL for each 6-well plate harvested.
Skip to step 26 below.
16. If Collagenase Solution was used, following incubation,
aspirate the Collagenase solution from each well, taking care
not to remove any floating colonies (see Note 5).
17. Add 1 mL of preferred KOSR containing medium to each well.
18. Using a 5 mL pipette, take up the medium from several wells,
and remove the cells from the plate by gently expelling while
moving the tip of the pipette back and forth across the colo-
nies. Colonies should readily detach. Take care not to disag-
gregate the colonies too much. Thawing efficiency will be
improved if larger colonies are maintained at freezing.
19. Pool the cells in a 50 mL conical tube.
20. Wash each plate with an additional 3 mL of preferred culture
medium, and add suspension to the pooled cells in the 50 mL
conical tube.
21. Centrifuge pooled cells at 200 × g, redistributing into smaller
pools if necessary.
22. Aspirate supernatant, and resuspend cell pellet in preferred
hPSC medium equal to 3 mL for each 6-well plate harvested.
23. Gently mix pooled cell suspension by pipetting up and down,
taking care not to further disaggregate the colonies.
24. Transfer 9 mL of cell suspension to a new 50 mL conical tube.
25. Add 9 mL of the appropriate cryopreservation medium to the
pooled cells dropwise, slowly, while gently tapping the side of
the tube to mix.
(d) If preferred culture medium is TeSR based, TeSR
Cryopreservation Medium should be used.
(e) If preferred hESC culture medium is E8 based, E8
(f) If preferred hESC culture medium is KOSR based, KOSR
26. At this point, the cells are in contact with DMSO, and work
must be performed as quickly as possible. Once cells are in
contact with DMSO they should be in the freezer within
2–3 min.
27. Pipette gently to mix cell suspension, being careful not to
further disaggregate colonies.
28. Using a 5 mL pipette, take up 6 mL of the prepared suspension,
and add 1 mL to five of the prepared cryovials, being careful
not to touch the pipette tip to the cryovial. Return the final mL
to the cell pool and repeat until the cell pool with cryopreser-
vation medium is exhausted (18 vials).
29. Place the lids on the cryovials, and ensure that they are tight
and secure.
30. Quickly place the cryovials into the room temperature Mr.
Frosty or CoolCell container, and place the container in the
−80 °C freezer overnight. Repeat steps 23–29 until the pooled
cell suspension is exhausted.
31. Twenty-four hours later, remove the frozen cryovials from
containers in the −80 °C freezer, and place into long-term LN2
storage.
3.2.4 Thawing Slow It is absolutely essential that safety glasses be worn during this pro-
Frozen hPSCs cedure, as while it is a rare occurrence, sealed vials can explode
during the thaw process.
1. Pre-warm a prepared 6-well cell culture plate using the same
culture platform (media and matrix) that the cells were cultured
in prior to cryopreservation. In general for TeSR and E8-based
cultures, a single frozen vial can be thawed into three wells of
a 6-well plate. KOSR-based cultures should be thawed into
one well of a 6-well plate.
2. Retrieve the vial to be thawed from long-term storage and
transport to the lab in a dewar filled with LN2.
3. Confirm the cap is tightly closed, and using metal forceps,
immerse the vial in a 37 °C water bath without submerging
the cap. Swirl the vial gently.
4. When only a small ice crystal remains, remove the vial from the
water bath.
5. Confirm again that the vial cap is tightened, and immerse the
vial into 95% ethanol.
6. In the biosafety cabinet, open the vial being careful not to
touch the rim of the vial.
7. Using a sterile 1 mL pipette, remove the cell suspension and
gently transfer into a sterile 15 mL conical tube.
8. Slowly add 5–10 mL of appropriate culture medium dropwise
to the cells in the 15 mL conical tube, gently moving the tube
back and forth to mix. It is critical to add the medium slowly
to avoid osmotic shock to the cells.
9. Centrifuge at 200 × g for 5 min.
10. Aspirate the supernatant, being careful not to disturb the cell
pellet (see Note 8).
11. Resuspend the cell pellet in culture medium so that there will
be a final volume of 2 mL per well total in the prepared culture
plate after adding the cells (i.e., if plates were prepared with
1 mL of media per well, and the vial will be thawed into three
wells, resuspend in 3 mL medium, ultimately adding 1 mL of
suspension to the 1 mL in the plate for a total of 2 mL).
12. Slowly add the cell suspension dropwise to the prepared plate
for recovery and expansion.
13. Place plate gently into the incubator. Distribute the cells by
gently shaking the plate front to back, rest a few seconds, then
side to side, and close the incubator door gently.
3.3 Controlled Rate The method described below was primarily developed for cGLP/
Freezing Method cGMP application toward clinical compliance [4, 5]. Whatever the
level of quality assurance, it is suitable for routine application, afford-
ing high-throughput and post-thaw cell viability (i.e., typically yield-
ing approximately 80–90% survival). Moreover, it provides for
high-throughput processing for small- or large-scale cell banking.
3.3.1 Controlled Rate 2. Program the biological control rate freezer according to Table 1.
Freezing 3. Pre-warm DPBS (with Ca2+ and Mg2+) to 37 °C and equilibrate
preferred hPSC culture media to 37 °C and 5% CO2.
4. Dilute Collagenase IV 1:20 in DPBS (with Ca2+ and Mg2+) for
a working solution of 1 mg/mL.
5. Aspirate hPSC culture media from culture vessels and rinse
cells three times with DPBS (with Ca2+ and Mg2+).
6. Add Collagenase IV working solution to culture vessels (e.g.,
0.5 mL per organ culture dish or 1 mL per T-25 flask) and
incubate at 37 °C in 5% CO2 for 8–10 min (see Note 6).
7. If using organ culture dishes flush and detach colony fragments
and cells from the substratum using a P200 pipettor. If using a
flask, then use a policeman to gently scrape the colonies from
the flask (see Note 7).
8. Transfer fragments to a 15 mL tube with 6 mL 37 °C hPSC
culture media and further disaggregate using a motorized 5 mL
pipette; for a 5 mm diameter hPSC colony, aim to generate
eight to ten evenly sized fragments.
9. Add at least five volumes of 37 °C hPSC culture media (i.e., 5×
the volume of Collagenase working solution).
10. Centrifuge at 300 × g for 3 min, remove supernatant, and
resuspend cells in CryoStor™ CS10 solution (cryo-media).
The volume of cryo-media depends on the number of straws
to be prepared, with 0.2 mL required per straw.
11. Maintain cells in cryo-media at 4 °C (e.g., on ice) for 10 min.
12. Transfer 0.2 mL of cells in cryo-media to a 0.5 mL CBS™
High Security Straw using a P200 pipettor. The straw must be
held at the top, where the safety stopper ensures sterility is
maintained throughout the filling procedure. The open end of
the straw must be protected from any contamination.
13. The open ends of the straw should be placed in the jaws of a
sealing unit, and each sealed hermetically. If necessary label each
straw for future identification.
Table 1
Control rate freezing program for hPSCs
Rate
Step (Ramp) From (°C) To (°C) (oC/min) Time (min) Action
1. 4 4 0 5 Hold
2. 4 −8 −1.0 Cold activation
3. −8 −8 0 10 Soak
4. −8 −8 0 Manual seeding
5. −8 −8 0 5 Hold
6. −8 −38 −0.8 Dehydration
7. −38 −100 −10.0 Rapid cool
8. −100 −180 −35.0 Plunge
9. −180 Hold
14. Maintain the straws at 4 °C (e.g., on ice) until all have been
sealed.
15. Ensuring the biological control rate freezer has reached 4 °C,
load the straws into the freezing chamber.
16. Select Run from the main menu to commence freezing.
17. For manual ice nucleation, seed at −8 °C, after soaking the
cells at −8 °C for 5 min. Ice-nucleate the cells by contacting
LN2 cooled forceps to the straws in the region of the media/
cell column.
18. Press enter to resume the profile.
19. With completion of a run (−180 °C), quickly remove the
straws avoiding thawing of contained cells and plunge to LN2.
20. Store in long-term LN2 storage.
3.3.2 Thawing Control 1. Quickly transfer frozen straw(s) from LN2 storage canisters to
Rate Frozen hPSCs a holding container filled with LN2. Avoid thawing of frozen
cells.
2. Immediately thaw a straw by gently swirling for 10–20 s in 37 °C
dH2O until a small ice pellet remains.
3. Completely plunge the straw in 95% ethanol and dry.
4. Once dry, cut off the end of the straw about 1 cm from the
hydrophobic plug, using a sterile scalpel or a pair of scissors.
5. Similarly cut off the other end of the straw proximal to the seal.
6. Using a P100 pipettor, empty the straw into a 14 mL conical
centrifuge tube with 6 mL warm hPSC culture media. Expel
the straw contents by placing the pipette tip in the top end of
the straw and ejecting.
7. Rinse the straw with additional 0.5 mL hPSC culture media.
8. Centrifuge cells at 300 × g for 3 min.
9. Aspirate supernatant from pelleted cells and resuspend in
0.5 mL hPSC culture media.
10. Transfer the cell suspension to prepared culture vessels for recovery
and expansion.
4 Notes
1. The Slow Freezing Method cryopreservation protocol may be

effectively used with a variety of culture platforms, including
mTeSR1 or TeSR2 with Matrigel or Vitronectin, TeSR-E8 or
Essential 8 Media with Matrigel or Vitronectin, or standard
KOSR-based hESC media on MEF or Matrigel platforms.
The culture platform used in cell expansion will impact the
appropriate choice for harvesting cells for cryopreservation to
achieve best results, as well as the components of the cryo-
preservation medium.
2. For microdissection, cutting pipettes can be obtained from a
commercial source or produced in-house from glass capillaries
heated in a Bunsen burner flame and pulled and broken to
~0.25–1.0 mm diameter. Over-pulling will result in a cutting
tip that is too fine and breaks during cutting. A blunt tip will
result in tearing rather than cutting of a colony.
3. DMSO should never pass through a filter. If it is necessary to
filter sterilize the other components of the cryopreservation
medium, do so before adding DMSO.
4. When labeling vials for freezing, the passage number should
be increased by one when compared to the animated culture
being frozen. This allows the culture vessel to be labeled at
thaw to match the vial being thawed and maintain passage
accuracy.
5. If a significant number of colonies are floating, scrape the cells
into the dispase solution rather than aspirating it. If this is done,
an additional spin wash with DMEM/F12 will be necessary.
6. While there are a variety of methods for enzymatic digestion,
here we describe a Collagenase IV-based approach. Alternatively,
Trypsin/EDTA can be employed although in our experience
hPSCs are more sensitive to Trypsin/EDTA compared to
Collagenase IV.
7. Cell scraping reduces the time of harvest. Alternatively, spon-
taneous detachment of colonies without scraping can be
achieved through extended exposure of cultures to Collagenase

IV for 30 min. The latter is compatible with closed culture
platforms such as cell factories.
8. Vacuum aspiration should be used with extreme caution, as
there is a significant risk of aspirating the cell pellet. If the
pellet is smaller than anticipated, or if the cells are particularly
valuable or rare, use a sterile serological pipette to remove the
supernatant.
References
1. Reubinoff BE, Pera MF, Vajta G, Trounson A 3. Crook JM, Peura TT, Kravets L et al (2007) The
(2002) Effective cryopreservation of human generation of six clinical grade human embryonic
embryonic stem cells by the open pulled stem cell lines. Cell Stem Cell 1:490–494
straw vitrification method. Hum Reprod 16: 4. Rall WF (2003) Avoidance of microbial cross-
2187–2194 contamination of cryopreserved gametes,
2. Vajta G, Holm P, Kuwayama M, Booth PJ, embryos, cells and tissues during storage in liquid
Jacobsen H, Greve T, Callesen H (1998) Open nitrogen. The Embryologists’ 6:4–15
pulled straw (OPS) vitrification: a new way to 5. Crook JM and Kravets L (2005) Cell preserva-
reduce cryoinjuries of bovine ova and embryos. tion method. Patent International Publication
Mol Reprod Dev 51:53–58 Number WO2005/118785 A1
Chapter 12
Genome Editing in Human Pluripotent Stem Cells

Jared Carlson-Stevermer and Krishanu Saha
Abstract
Genome editing in human pluripotent stem cells (hPSCs) enables the generation of reporter lines and
knockout cell lines. Zinc finger nucleases, transcription activator-like effector nucleases (TALENs), and
CRISPR/Cas9 technology have recently increased the efficiency of proper gene editing by creating double
strand breaks (DSB) at defined sequences in the human genome. These systems typically use plasmids to
transiently transcribe nucleases within the cell. Here, we describe the process for preparing hPSCs for
transient expression of nucleases via electroporation and subsequent analysis to create genetically modified
stem cell lines.
Key words Genome editing, Genomics, CRISPR/Cas9, Electroporation
1 Introduction
Nuclease-based gene editing relies on endogenous DNA damage

repair processes to edit desired sequences in the genome. Double
strand breaks generated by nucleases are typically repaired via one
of two processes: homology-directed recombination (HDR) and
non-homologous end joining (NHEJ) [1]. HDR using donor
DNA templates or single-stranded oligos (ssODNs) results in
error-free incorporation of foreign DNA sequences. NHEJ, in
contrast, generates a variety of insertions and deletions at the DSB
site, which in some cases results in frameshifts causing premature
stop codons to generate knockout cell lines.
Currently, the most common method to generate gene-edited
hPSCs uses a clustered regularly interspaced palindromic repeat
(CRISPR)/CRISPR-associated protein (Cas9) system [2].
Engineering of the type II bacterial CRISPR-associated (Cas)
system into human cells [2] has dramatically increased the rate of
which genetic engineering has progressed. Cas9 is an RNA-guided
endonuclease that uses short guide RNAs (gRNAs) to selectively
create DSBs at any point in the genome that is flanked by a 3′
protospacer adjacent motif (PAM). When DSBs are created in cells,
165
166 Jared Carlson-Stevermer and Krishanu Saha
Fig. 1 Electroporation of Cas9 plasmid and AAVS1 T2 gRNA plasmid (Addgene

#41818), and GFP donor plasmid (Addgene #28014) yielded targeted GFP cell
lines. This example uses HDR targeting techniques
endogenous repair mechanisms are activated, resulting in repair

from a donor via HDR or NHEJ. This method of gene editing was
previously shown using zinc-finger nucleases [3] and TALENs [4]
in hPSCs. HDR-mediated insertion of transgenes occurs naturally
in hPSCs [5], but occurs at a much higher rate when DSBs are
introduced. Transgenes can be stably inserted into the genome by
HDR if a donor is supplied that contains the transgene surrounded
by regions homologous to the area where it should be inserted.
Single nucleotide polymorphisms (SNP) can also be introduced by
transfecting with a ssODN that is homologous to the region
around the cut site but for the point mutation of interest [3, 6].
Three methods in which the necessary components can be
introduced into the cell include: (1) transient transfection of two
plasmids, one containing the coding sequence for Cas9 and the
other expressing the gRNA [2, 6], (2) viral integration of the Cas9
coding sequence and transient expression of the gRNA [7], and
(3) viral integration of both Cas9 and gRNA sequences [8–10].
Delivery of nucleic acids to hPSCs is typically limited using stan-
dard lipofection reagents, so most studies utilize electroporation or
nucleofection [11]. Efficiency of the two plasmid system has been
increased by utilizing a Cas9-2A-GFP plasmid [12] that can be
used to enrich the cell population expressing Cas9 via fluorescence-
activated cell sorting (FACS) of GFP-expressing cells. Using these
strategies, we have been able to insert transgenes (Fig. 1) [13] and
detect frameshifting indels causing a knockout phenotype in more
than 25% of cultured hPSCs.
2 Materials
1. Dulbecco’s Phosphate-Buffered Saline, without calcium,

without magnesium (DPBS, Life Technologies).
2. mTeSR1 media (STEMCELL Technologies).
Genome Editing in Human Pluripotent Stem Cells 167
3. Y-27632 (ROCK Inhibitor, STEMCELL Technologies).

4. Cas9-2A-GFP Plasmid (Addgene #44719).
5. gRNA vector (Addgene #41824).
6. DMEM/F12 (Life Technologies).
7. Accutase (STEMCELL Technologies).
8. Microcentrifuge Tubes (Fischer Scientific or other sources).
9. 15 mL Conical Tubes (BD Biosciences or other sources).
10. Standard micropipettes with P2.5, P20, P200, P1000 + tips.
11. Hemocytometer (Hausser Scientific or other sources).
12. Bio-Rad cuvette, 0.4 cm (1 per transfection).
13. Matrigel®-coated tissue culture plates (Corning).
14. Electroporator (Bio-Rad or equivalent for mammalian cells).
15. QuickExtract (Epicenter).
16. AccuPrime Taq High Fidelity (Life Technologies).
17. Forward and reverse primers for genomic locus (IDT or other
DNA synthesis vendors).
3 Methods
3.1 Transfection 1. Seed human PSCs at a density such that they are about 70–80%
Protocol confluent in 2–3 days (see Note 1).
3.1.1 Setup 2. Twenty-four hours prior to transfection, change media to
include 10 μM ROCK Inhibitor (see Note 2).
3. Prepare Matrigel® plates for subsequent culture of electroporated
cells. Cells may be plated at a density of 104 to 105 cells per cm2.
4. Prepare plasmid stocks for electroporation (see Note 3).
5. Warm Accutase and cell culture reagents to 37 °C prior to
starting experiments.
3.1.2 Experimental 1. Prepare electroporation solution containing plasmids and media.

(a) Determine plasmid volume for each plasmid. 2.5 μg of
Cas9 plasmid and 1.0 μg of gRNA plasmid are needed for
every 1 × 106 cells to be transfected (see Note 4). If using
a donor DNA template, add 2.0 μg per 1 × 106 cells. If
using a ssODN template, add 1 μL of a 10 μM stock
(see Note 5) [6]. For protocol modifications use ZFN and
TALEN strategies (see Note 6).
(b) Pipet 400 μL mTeSR1 media into a microcentrifuge tube.
(c) Thaw and gently vortex plasmid stocks.
(d) Add plasmids to media.
(e) Plasmid solution should be kept on ice.
2. Aspirate media from the human PSC wells and wash with
1–2 mL of DPBS.
3. Harvest cells via Accutase enzymatic detachment.
(a) Add enough Accutase to cover well and place in a 37 °C
incubator for 5–7 min.
(b) Dislodge cells by tapping the culture plate, then collect
cells from each well using a P1000 pipette and add cell
suspension to a 15 mL conical tube.
(c) Gently pipet up and down to dissociate any remaining
clumps (see Note 7).
(d) Immediately rinse each well with an additional 0.5 vol-
umes of culture media or DMEM/F12 and add to the
conical tube to collect any remaining cells and neutralize
Accutase by dilution.
4. Obtain cell count via hemocytometer or flow cytometry-based
methods.
5. Remove sufficient volume to obtain the desired cell count per
transfection (0.5–5 × 106 per transfection has been shown to
be effective) and place into a 15 mL conical tube.
6. Pellet cells by centrifuging 5 min at 200 × g.
7. Aspirate supernatant and resuspend pellet in the cold electropor-
ation solution created in Subheading 2, item 1 (see Note 8).
8. Transfer solution into a 0.4 cm cuvette (see Notes 9 and 10).
9. Pulse cuvette in electroporator. High efficiency has been
observed using an exponential decay waveform, 250 V, 750 μF
capacitance, and infinite resistance (see Notes 11–13).
10. Using a P200 pipette, remove electroporation solution from
cuvette and transfer into a 15 mL conical tube, containing
sufficient volume of mTeSR + 10 μM ROCK Inhibitor medium
for desired number of wells (see Note 14).
11. Wash cuvette with a 200 μL aliquot of mTeSR media to remove
any remaining cells, add wash to the 15 mL conical tube.
12. Plate cells into culture dish.
13. At 24 h post-electroporation, change media with mTeSR1
+ 10 μM ROCK Inhibitor.
14. At 48 h post-electroporation, ROCK Inhibitor may be removed
and selection agents (if any) may be added.
3.1.3 Assaying 1. Allow cells to grow to ~80% confluency.

the Efficiency 2. Passage wells at a 1:2 split ratio.
of NHEJ-Mediated Indels
3. Allow to grow until all wells are again ~80% confluent with
daily media changes of mTeSR without ROCK inhibitor.
Fig. 2 Representative image from 2 T7 assays done in parallel. Each sample is

from a different gRNA targeted against a mCherry transgene. Three lanes of each
genomic PCR product are run. Primers: Forward: AAGGGCGAGGAGGATAACATGG;
Reverse: TTGTACAGCTCGTCCATGCCG
4. Harvest genomic DNA using QuickExtract solution or equivalent

extraction method (see Note 15).
Run one of the following assays for the detection of NHEJ: T7
Endonuclease (Fig. 2) or SURVEYOR [14] (see Note 16), PAGE
gel [15], MiSeq [16], of HRMA [17]. Mutational frequencies can
vary between assays, as do the lower limits of resolution for each
assay. T7 Endonuclease assays require ~5% NHEJ to be resolved,
whereas MiSeq can detect a single cell in which a mutation has
occurred. The sensitivity of other assays falls between these two
extremes.
3.1.4 Isolating Clonal 1. If you need to schedule an appointment at a FACS sorting

Populations via FACS facility, schedule a time 2–3 days after electroporation.
Sorting 2. Follow electroporation protocol. In Subheading 2, item 1,
substitute Cas9 plasmid with Cas9-2A-GFP plasmid.
Alternatively, in Subheading 2, item 1, add 250 ng of GFP
plasmid per 1 × 106 cells (Addgene #13031).
3. At 24–48 h post-electroporation, confirm transfection success
using fluorescent microscopy.
4. Add 10 μM ROCK Inhibitor to transfected wells 1 h prior to
dissociation (see Note 17).
5. Obtain a single-cell dissociation using Accutase as described in
Subheading 2, item 3.
6. Centrifuge for 5 min at 200 × g.
7. Resuspend in 2–3 mL of mTeSR media + 10 μM ROCK inhibitor
and pass through a 40 μm cell strainer cap into a FACS tube.
8. Keep cells and collection tubes on ice (see Note 18).
9. Using FACS, collect cells that are expressing GFP. Ideally, use
cells that were mock electroporated (no plasmid) to set up
gates for fluorescence (Fig. 3).
a b c
GFP- , mCherry+ GFP+ , mCherry+ GFP- , mCherry+ GFP+ , mCherry+ GFP- , mCherry+ GFP+ , mCherry+
105 4.24E-3 0 105 98.8 0.12 105 67.5 30.3
mCherry Fluorescence (RFU)
104 104 104
103 103 103
0 0 0
GFP- mCherry- GFP+, mCherry- GFP- mCherry- GFP+, mCherry- GFP- mCherry- GFP+, mCherry-
-103 99.9 0.076 -103 1.10 8.30E-3 -103 0.88 1.29
-103 0 103 104 105 -103 0 103 104 105 -103 0 103 104 105
GFP Fluorescence (RFU) GFP Fluorescence (RFU) GFP Fluorescence (RFU)
Fig. 3 Representative flow cytometry plot for sorting Cas9- and GFP- expressing hPSCs. (a) Non-fluorescent
hPSC-negative control. (b) Mock electroporated H9-H2B-mCherry expressing cells. (c) Cas9, eGFP-N1 co-
electroporated hPSCs. This method shows >30% efficiency of introduction into hPSCs
10. Transfer cells into fresh wells of a 6-well plate at a density of

~1000 cells/cm2 in mTeSR1 with 10 μM ROCK inhibitor.
11. Allow cells to grow for 7–10 days while performing daily media
changes with mTeSR1 with 10 μM ROCK inhibitor (see Note
19). Other cloning methods can also be successful (see Note 20).
12. When colonies begin to form, use a P200 pipette to lightly
scrape at colonies of interest while slowly releasing the plunger
(see Note 21).
13. Transfer each clone to a single well of a 24-well plate.
14. When each clone is individually confluent (typically 4–5 days
after previous step), passage at a 1:3 ratio into three wells of a
12-well plate.
15. From one well, harvest genomic DNA using QuickExtract
solution (see Note 15) or equivalent extraction method.
16. Individually freeze other two wells (see Chapter 13) in LN2.
17. Prepare harvested genomic DNA for Sanger sequencing [18].
After receiving sequencing results, thaw and culture clones
that contain desired mutations (see Chapters 11 and 13).
4 Notes
1. Cells must be in an actively dividing state.

2. The longer the cells are in ROCK Inhibitor, the greater the cell
attachment following electroporation (18–24 h is recom-
mended). The minimum amount of time for cells to be in
ROCK inhibitor that has an appreciable effect is 1 h.
3. Concentration will depend on efficiency of plasmid isolation

protocol. Ideal values will be greater than 1 μg/μL as determined
by NanoDrop or equivalent method. Plasmid purification
protocol must contain an endotoxin removal step.
4. An alternative to transfecting plasmids is to directly transfect
the RNA and Cas9 protein required. The advantage of this
alternative is that there is less of a lag time until results can be
collected. In this alternative, generate gRNAs via in vitro
transcription [7, 19] off of the created gRNA plasmid using
the MEGAshortscript T7 kit (Ambion). Cas9 coding
sequence can be cloned into a bacterial expression vector and
expressed in a bacterial host before being purified via an
attached His tag [20].
5. Optimal ssODN design is necessary for successful HDR. Some
of the considerations that must be taken into account are the
mutation of the PAM sequence via codon wobble to produce
a silent mutation [21]. Without this modification, the Cas9-
gRNA complex will cleave the ssODN. Second, design
intended point mutations in the center of ssODN with ~40 bp
flanking homology on either side [22]. It is also advisable to
design your ssODN to either introduce or remove a restriction
enzyme site [23]. This consideration will allow for facile analysis
of clones, without having to perform one of the NHEJ assays
mentioned in Subheading 3.1.3.
6. Both zinc finger nucleases (ZFNs) and TALENs remain viable
options for gene editing. If using an either method, electro-
porate using 0.5 μg of nuclease encoding plasmid and 4 μg of
donor DNA per 1 × 106 cells [4, 24]. All other steps of the
protocol remain the same.
7. Single-cell dissociations are important as electroporation of
cell clumps decreases efficiency.
8. It is important to remove as much supernatant as possible, as
excess volume during electroporation will decrease efficiency.
9. Bubbles must be avoided, as they will cause increased cell death
during electroporation.
10. Avoid touching the metal sides of cuvette as oils may transfer
and cause waveform to be disrupted.
11. Individual machines vary, so these values will have to be opti-
mized on a per-machine basis. High-voltage electroporations
will increase efficiency while also increasing cell death. We rec-
ommend starting at our settings at changing the voltage in
increments of ±25 V while holding the capacitance constant.
Then increment capacitance ±50 μF at optimum voltage.
12. Make a note of time constant (TC) values. Highly variable values
may indicate a malfunctioning machine.
13. Variability between stem cell lines, especially between embryonic

stem cells (ESCs) and induced pluripotent stem cells (iPSCs),
can have a drastic effect on cell viability. If you are experiencing
high amounts of cell death, go back and reoptimize electropora-
tion conditions for each cell line. This is especially necessary if
transitioning conditions from an ESC line to an iPSC line.
14. We generally electroporate enough cells to provide six repli-
cates in a full 6-well plate. Using this setup 12 mL of mTeSR
would be needed per electroporation.
15. To perform an extraction using QuickExtract, cells are first
trypsinized, pelleted, and resuspended in 500 μL of
QuickExtract before being transferred into a microcentrifuge
tube. Then place microcentrifuge tube in a heat block at 65 °C
for 15 min, 68 °C for 15 min, and 98 °C for 10 min.
16. SURVEYOR from Transgenomic is similar to the T7
Endonuclease assay. We have found that reagents can be
exchanged between the two kits.
17. ROCK inhibitor may be left in culture from pre-
electroporation through FACS without decrease in viability
or proliferation rate.
18. Sorting cells into collection tubes may be beneficial to sorting
directly into culture plates. Sorting into plates causes cell death
due to cells slamming into the plastic on the plate bottom.
Sorting parameters need also to be modified for human PSCs.
For a BD Aria, we use a nozzle size of 100 μm and a pressure of
20 psi.
19. Do not allow colonies to grow too large or merge.
20. Single-cell cloning can also be done in a high-throughput
method if mutations happen at a frequency of <1%. This
method, known as sib-cloning, combines a TaqMan PCR assay
with fluorescent probes that distinguish between mutant and
wild-type products. Mutant frequencies as low as 0.1% can be
detected and enriched by selection and propagation to a further
round of cloning [25].
21. Be sure to remove entire colony from culture plate. If cells are
detached they may float through media and contaminate other
clones being isolated. To avoid this contamination, change
media between each clone being selected.
FACS sorting at clonal density culture can survive without
differentiation 48–72 h without changing media. This simu-
lates the use of conditioned media and decreases stress on sin-
gle cells. After this period, change media daily. When colonies
form after 7–10 days use a P200 pipette set to 200 μL and,
with the plunger depressed, gently scrape at the colony edge
causing it to lift off the plate. Simultaneously, slowly release the
plunger to suck the entire colony into pipette tip. Transfer into
new well and gently pipet up and down three to four times to
dissociate the colony. This dissociation can dramatically speed
up the rate of proliferation. Change media in new well every
24 h. Wait until cells are ~80% confluent, usually about
4 days, and then passage 1:3 into three wells. Wait another
4–5 days or until wells are again ~80% confluent before freez-
ing two wells. Two wells are frozen to provide a backup in the
event one sample does not survive the freeze/thaw cycle.
Collected genomic DNA from final well should be PCR ampli-
fied using AccuPrime Taq with primers for the locus of interest
and then Sanger sequenced using the same forward primer as
used for amplification.
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Part III
Protocols for Mesenchymal Stem Cell Banking

Chapter 13
Isolation, Culture, and Expansion of Mesenchymal

Stem Cells
Izaskun Ferrin, Izaskun Beloqui, Lorea Zabaleta, Juan M. Salcedo,
Cesar Trigueros, and Angel G. Martin
Abstract
Mesenchymal stem cells (MSCs), together with hematopoietic stem cells (HSCs), are the most frequently
used cell type for cell-based therapeutics. As for other cell types intended for research and translational use,
it is important to establish correctly typed cell lines from human tissue donations. Here, we describe
methods for isolating, culturing, and identifying MSCs from various tissues obtained through human tissue
donation. The methods have been used in the context of a biobank, prepared as standard operating
procedures (SOPs), ensuring traceability and reproducibility of cell production.
Key words Bone marrow, Umbilical cord, Lipoaspirate, Dental pulp, Mesenchymal stem cells
1 Introduction
Regenerative medicine research has gained extraordinary momen-

tum in the last 10 years, mainly due to advances in the stem cell
field. However, cell therapy has been realized in just a handful of
clinical indications, but nonetheless promises to provide novel
solutions to currently untreatable conditions. Cell therapy is largely
based on the use of stem cells as therapeutic elements to promote
tissue regeneration, cell replacement, or immunoregulation. MSCs,
also known as mesenchymal stromal cells, originally described in
bone marrow [1], are the most widely recognized cell type for
clinical use for several reasons: they are easy to obtain and cultivate
in big numbers, are capable of multilineage differentiation, have
the potential for autologous or allogeneic use, have immunoregu-
latory features, and present few ethical dilemmas (with IRB
approval and donor consent). MSCs can be isolated with high
efficiency from various tissues, such as bone marrow [2], adipose
tissue [3], umbilical cord [4], and deciduous teeth [5]. The fre-
quency of MSCs in the tissues ranges from 0.001% of nucleated
177
178 Izaskun Ferrin et al.
cells in bone marrow to 0.3% in the perivascular compartment sur-

rounding the vessels of the umbilical cord [6] to 2% in the vascular
stromal fraction of adipose tissue [7]. MSCs have been shown to
differentiate into mesodermal lineages (such as bone, fat, or carti-
lage) [8], and have the ability to inhibit T-cell proliferation and
activation, which together with their low immunogenicity suggests
that they may have potential for allogeneic application [9]. Unlike
hematopoietic precursors, plastic-adherent MSCs can be expanded
in culture without loss of differentiation potential, although their
lifespan is limited to 15–50 population doublings [10]. Moreover,
MSCs lack a set of unique surface markers for identification, which
minimally relies on the expression of several stromal markers and
the ability to differentiate in vitro in response to particular stimuli
[11].
There are currently more than 200 active clinical trials using
MSCs. In order to maximize their therapeutic potential, a thor-
ough understanding of MSC biology is required, necessitating
high quality and characterized cell stocks. Antecedent to quality
biobanking, here we describe reliable and standardized procedures
for isolating, culturing, and phenotyping human MSCs from bone
marrow, umbilical cord wall (Wharton’s jelly), adipose tissue, and
dental pulp.
2 Materials
2.1 Consumables 1. Tissue culture ware: T175 flasks, T25 flasks, 6-well plates.
and Equipment 2. Automatic pipettor.
3. Sterile pipettes (plastic).
4. Sterile Pasteur pipettes.
5. Sterile tubes with conical bottom 50 mL (Becton-Dickinson;
Franklin Lakes, NJ; ref.: 352,098).
6. 1.5 mL eppendorf tubes.
7. Sterile blades.
8. Scissors.
9. Nylon sterile cell strainers (40 and 70 μM).
2.2 Reagents 1. DMEM: Dulbecco’s modified Eagle’s Medium-Low glucose

(Sigma; St. Louis, MO).
2. Fetal Bovine Serum (FBS). FBS must be heat-inactivated
previous to use. To incativate, place in a tempered water bath
at 56 °C for 30 min.
3. 100X L-Glutamine solution.
Isolation, Culture, and Expansion of Mesenchymal Stem Cells 179
4. Culture medium: DMEM + 10% FBS + 1× L-glutamine.

5. Ficoll-Paque or Lymphoprep®.
6. Phosphate buffered saline—1× PBS- pH 7.2 (Invitrogen;
Carlsbad, CA).
7. Trypsin-EDTA solution 0.25%, (Sigma; St. Louis, MO; Ref:
T4049).
8. Adipocyte differentiation medium: culture medium supple-
mented with 1 μM dexamethasone, 500 μM 3-isobutyl-1-
methylxantine (IBMX), and 200 μM indomethacin.
9. Osteogenic medium: culture medium supplemented with
0.1 μM dexamethasone 50 μM ascorbic acid and 10 mM
β-glycerophosphate.
10. 6× Collagenase I solution: dissolve 1 g of collagenase type I
powder (Sigma; St. Louis, MO) into 22 mL of 1× PBS. Filter
sterilize through a 0.22 μM filter. Complete to 200 mL final
volume with 1× PBS.
11. Collagenase (3 mg/mL)-Dispase (4 mg/mL) solution (Roche;
Indianapolis, IN).
12. PBS staining buffer: PBS 1× + 1% BSA (Sigma; St. Louis, MO;
Ref: A9647) + 1% FBS + 0.05% Sodium Azide.
13. Anti-human CD-XX labeled primary antibodies, available from
several manufacturers. Use appropriate normal serum controls
according to the xenotype chosen for each marker.
14. 0.5% Oil Red O solution (Sigma; St. Louis, MO; Ref: O1391).
15. Paraformaldehyde solution (Stock): 1. Measure 100 mL
Phosphate Buffered Saline (PBS) into a measuring cylinder.
Pour into the conical flask containing 4 g of paraformaldehyde.
Cover with parafilm and transfer to the fume hood: thoroughly
shake—take care not to splash paraformaldehyde about—it is a
rapid fixer and is TOXIC. Place flask on the top of the hot-
plate/stirrer inside the fume cupboard and set the heat control
to 7 with moderate stirring. Allow the solution to warm up—it
will turn from being cloudy to clear when ready. Inspect regu-
larly to avoid over-heating and consequent spilling. When the
paraformaldehyde has dissolved, switch off the heat but leave
to stir: do not handle for safety reasons. Allow to cool. When
cooled, transfer the fixative to a 4C refrigerator. Label appro-
priately and date.
16. 70% Ethanol.
17. Alizarin Red S (Sigma; St. Louis, MO; Ref: A5533).
18. 10% Acetic acid.
3 Methods
3.1 Initial Sample The sample is a clinical bone marrow aspirate collected (in accor-
Processing dance with local safety regulations for handling uncharacterized
and Establishment blood samples) into standard blood collection tubes or bags con-
of Primary Cultures taining anticoagulant to prevent sample clotting. Sample volume is
usually in the order of 10–20 mL, containing typically 10–50 × 106
3.1.1 Bone Marrow Bone Marrow Mononuclear Cells (BMMCs); once drawn, storing
the blood sample at room temperature should not exceed 4 h
(see Note 1).
1. Spray the tubes with 70% ethanol before placing them in the
laminar flow hood to ensure loss of external contaminants.
2. Carefully remove the sample and place it in a 50 mL tube.
3. Dilute the sample with 1× PBS to a final dilution 1/4.
4. Load onto a Ficoll-Paque density gradient in sterile 50 mL
conical tubes containing at least 1/3 volume of Ficoll-Paque
(see Note 2).
5. Centrifuge at 800 × g (1800–2000 rpm in a conventional bench
top centrifuge) for 20 min at room temperature, without brake
(rotor braking would distort the gradient interphase).
6. Collect the interphase containing the mononuclear cells into a
labeled tube (see Note 3). Collect a sample of serum and store
at −20 °C for viral pathogen testing.
7. Remove the excess of Ficoll-Paque by adding two volumes of
1× PBS; centrifuge at 400 × g for 10 min.
8. Carefully discard the supernatant and resuspend the BMMCs
in 3 mL of fresh culture medium by forcefully tapping until no
larger clumps can be seen.
9. Count the cells with a Neubauer hemacytometer, diluting
small aliquots of cells in trypan blue.
10. Plate remaining cells to a final cell concentration of 0.5–
1.5 × 106 cells per cm2 in appropriate tissue culture ware
(typically a 6-well plate).
11. Culture the cells for a period of 24 h in a 5% CO2 incubator
at 37 °C.
12. After 24 h carefully remove the culture medium using a sterile
pipette, collecting as many cells that remain in suspension as
possible.
13. Wash the cells attached to the plate once with 1× PBS.
14. Add fresh culture medium to the cells and incubate further
for 24 h.
15. Repeat steps 12–14, changing the medium every 3–4 days.
Remaining cells attached to the plate are enriched in MSCs.

This culture constitutes Passage 0.
16. When the culture reaches 80–90% confluence, cells should be
trypsinized and plated at 1000–2000 cells per cm2 for further
expansion of the culture.
17. For trypsinization, aspirate the culture medium with a vacuum
attached pipette, taking care not to disturb the adherent cells.
18. Rinse with 1× PBS to remove the excess medium. The volume
of 1× PBS used should equal the original medium volume.
Carefully remove the PBS 1×.
19. Add tempered Trypsin-EDTA to detach the cells, adding the
necessary volume to cover the entire cellular surface and
lightly shake flask. Place the flask in the incubator for not
more than 5 min.
20. Stop Trypsin-EDTA action adding temperate medium at a
1/3 dilution. Collect the mixture into a labeled centrifuge
tube. Using a microscope, confirm that there are no or few
cells left in the flask. Centrifuge at 400 × g for 5 min at room
temperature.
21. Remove the supernatant without disturbing the pellet. Carefully
resuspend the pellet with an appropriate volume (at least 5 volumes
of pellet size) of temperate culture medium.
22. Seed the cells at a final concentration of 1000–2000 cells per
square centimeter using appropriate plastic culture ware.
This constitutes Passage 1, which can now be frozen as Master
Cell Bank (MCB), or otherwise continue culturing to make
Working Cell Banks (WCB) (see Note 4).
3.1.2 Umbilical Cord The umbilical cord is typically discarded as medical waste post-
delivery, therefore making it a safe, noninvasive means to obtain
therapeutically beneficial stem cells. Wharton’s jelly is a mucous
connective tissue of the umbilical cord lying between the umbili-
cal vessels and the amniotic epithelium. First described by Thomas
Wharton in 1656, it prevents compression, torsion, and bending
of the umbilical vessels. Cells found in Wharton’s jelly are primitive
MSCs likely trapped in connective tissue as they migrate to the
mesonephros region in the developing cord, during embryogen-
esis [12].
All steps must be performed in a biosafety cabiner/laminar
flow hood.
1. Place the umbilical cord on a sterile metal tray. The umbilical
cord will come clamped at both ends from the delivery room.
2. Puncture the vein with a needle to withdraw a few milliliters of
blood.
3. Centrifuge the blood at 400 × g for 5 min.

4. Collect the blood serum and store at −20 °C for viral pathogen
testing.
5. Subsequently remove the rest of cord blood by removing the
clamp at one of the ends.
6. Wash umbilical cord extensively using 1× PBS and remove the
second clip. Repeat these washings until no traces of blood are
observed.
7. Dissect the umbilical cord into small pieces (3 mm3). Place
these fragments on the surface of 6-well plates. Fill the plate
until approximately half of the surface of each well is covered.
It is recommended to fill three plates (18 wells) to ensure suf-
ficient explant growth.
8. Incubate for 2 h at 37 °C to get the explants to adhere to the
plastic plate.
9. Add 3 mL of cell culture medium to each well, being careful
not to lift the explants (see Note 5).
10. Culture for a period of 24 h in a 5% CO2 incubator at 37 °C.
11. Remove the culture medium using a sterile pipette, trying to
collect all cells that remain in suspension.
12. Add fresh medium to the plates and return to the incubator.
13. After 3–5 days when cells are readily observed growing from
the explants, remove the explants by pulling them off the plate.
14. Change the culture medium every 3–4 days with fresh medium
until an 80–90% confluence is reached.
Cells attached to the plate are enriched in MSCs. This culture
constitutes Passage 0.
16. For trypsinization carefully aspirate the culture medium with
a vacuum attached pipette, taking care not to disturb the
adherent cells.
of PBS used should equal the original medium volume.
Carefully remove the PBS.
18. Add tempered Trypsin-EDTA to detach the cells adding the
more than 5 min.
19. Stop Trypsin-EDTA action adding temperate medium at a 1/3
dilution. Collect the mixture into a labeled centrifuge tube.
Using a microscope, confirm that there are no or few cells left in

the flask. Centrifuge at 400 × g for 5 min at room temperature.
resuspend the pellet with an appropriate volume (at least 5
volumes of pellet size) of temperate medium.
cm2 using appropriate plastic culture ware.
This constitutes Passage 1, which can now be frozen as a MCB,
or otherwise continue culturing to make WCBs (see Note 4).
3.1.3 Fat Aspirate Human adipose tissue, obtained by suction-assisted lipectomy

(i.e., liposuction), can be processed to obtain a fibroblast-like popu-
lation of cells, also known as a processed lipoaspirate (PLA).
Lipoaspirate from human subjects undergoing liposuction is deliv-
ered to the biobank in 50 mL tubes or syringes. Lipoaspirate typi-
cally yields a primary culture of 2 × 108 PLA cells per sample of
300 mL [3]. Freshly isolated PLA fraction contains a heterogeneous
population of endothelial, macrophages, and smooth muscle cells
(5–20%) and the rest (80%) are fibroblastoid cells of mesenchymal
origin. Contaminating cell populations decrease over time giving a
homogenous population similar in appearance to cultured MSCs
that can readily differentiate in vitro into adipogenic, osteogenic and
chondrogenic lineages upon treatment with lineage-specific factors.
1. Centrifuge the raw lipoaspirate at 450 × g for 5 min at room
temperature. After centrifugation three different fractions can
be observed: the top yellow fraction is fat; the lower fraction
is composed of the red cells and other contaminant blood
cells; the third fraction is the intermediate fraction. Discard
the top fraction and the lower fraction. Then collect the inter-
mediate fraction (stromal vascular fraction –SVF-) and use for
MSC isolation.
2. Wash the collected SVF extensively with equal volumes of 1×
PBS through three repeated cycles of centrifugation and super-
natant removal.
3. Incubate the washed SVFs aspirates with 1× collagenase type I
solution diluted in DMEM for 30 min at 37 °C with gentle
agitation.
4. Inactivate collagenase with a 1/10 volume of FBS and centri-
fuge for 5 min at 450 × g at room temperature. Remove the
supernatant.
5. Wash the pellet through three repeated cycles of centrifugation
with 5 volumes of 1× PBS and resuspend in 5 mL of DMEM
(low glucose) + 10% FBS.
6. Filter through a 40 μm nylon mesh to remove cellular debris.
7. Wash the filtrate through three repeated cycles of centrifuga-

tion with fresh medium, count the cells, and plate at a density
of 2000 cells/cm2.
These cultures represent the primary culture (Passage 0).
9. For trypsinization, carefully aspirate culture medium with a
vacuum attached pipette, taking care not to disturb the adher-
ent cells.
11. Add tempered Trypsin-EDTA to detach the cells, adding the
necessary volume to cover the entire cellular surface and lightly
shake the flask. Place the flask in the incubator for not more
than 5 min.
12. Stop Trypsin-EDTA action adding temperate cell culture
medium at a 1/3 dilution. Collect the mixture into a labeled
centrifuge tube. Using a microscope, confirm that there are no
or few cells left in the flask. Centrifuge at 400 × g for 5 min at
room temperature.
volumes of pellet size) of temperate culture medium.
3.1.4 Dental Pulp Human dental pulp stem/stromal cells (hDPSCs) possess gene
expression profiles and a differentiation capacity analogous to that
of bone marrow-derived mesenchymal stem cells (BMSCs) [13].
There are many reports addressing teeth regeneration with stem
cells from human dental pulp; therefore, banked hDPSCs have the
potential to be used for dental and medical treatment.
Normal human deciduous molars are collected as medical
waste from wisdom tooth removal. In the operating room, tooth
surfaces are cleaned and the surgeon cuts around the cementum-
enamel junction, using sterilized dental fissure burs to reveal the
pulp chamber. The pulp tissue is then gently separated from the
crown and root and collected in culture medium for transportation
to the biobank facility.
1. Digest the pulp material in a solution of Collagenase/Dispase

solution for 1–4 h at 37 °C with gentle agitation until com-
plete disaggregation occurs.
2. Filter through a 70 μm nylon mesh to remove cellular debris.
3. Inactivate collagenase with a 1/10 volume of FBS and then
centrifuge for 5 min at 450 × g at room temperature. Remove
the supernatant.
4. Wash the pellet through three repeated cycles of centrifugation
with 5 volumes of 1X PBS and resuspend in 5 mL of DMEM
(low glucose) + 10% FBS.
5. Wash the filtrate with fresh culture medium through three
repeated cycles of centrifugation, count the cells, and plate the
whole cell pellet in a p60 tissue culture plate.
6. The medium must be changed every 3–4 days until the cul-
ture achieves 80–90% confluency. Samples of the culture
medium should be analyzed for microbiological contamina-
tion (see Note 6).
These cultures represent the primary culture (Passage 0).
trypsinized and plated onto fresh culture ware at 1000–2000
cells per cm2 for further expansion of the culture.
8. For trypsinization, carefully aspirate the culture medium with
a vacuum attached pipette, taking care not to disturb the
adherent cells.
10. Add tempered Trypsin-EDTA to detach the cells adding the
more than 5 min.
11. Stop Trypsin-EDTA action adding temperate medium at a
1/3 dilution. Collect the mixture into a labeled centrifuge
tube. Using a microscope, confirm that there are no or few
cells left in the flask. Centrifuge at 400 × g for 5 min at room
temperature.
volumes of pellet size) of temperate culture medium.
3.2 Secondary After successfully establishing primary cultures and MCBs, the
Culture cultures may be further expanded to obtain WCBs. Before any
and Establishment further expansion, cultures must be tested for microbiological
of Working Stocks contaminants (bacteria, yeast, micoplasm, and viral pathogens).
Some biobanks may implement in house testing capacities for these
entities or otherwise outsource to a certified laboratory.
For cell passaging follow steps described above after establish-
ment of passage 0 (primary culture). We recommend expanding
MSC cultures up to passage 3 to ensure sufficient availability of
material while preserving the biological features of the original cul-
ture, thus preventing culture-associated adaptations. Figure 1
depicts a schematic representation of MSC isolation, referred to as
lipoaspirate (although generally applicable to bone marrow, umbil-
ical cord, or dental pulp).
3.3 Phentoyping The abbreviation MSC is used to represent bone marrow stromal
and Differentiation cells, mesenchymal stem cells, and multipotent mesenchymal stro-
mal cells [11], isolated, cultured, and characterized using different
methods. Importantly, the Mesenchymal and Tissue Stem Cell
Committee of the International Society for Cellular Therapy
(ISCT) have proposed minimal criteria for defining MSCs. First,
the cells should be designated mesenchymal stromal cells (MSCs).
MSCs must adhere to common plastic culture ware under standard
Fig. 1 Schematic representation of lipoaspirate MSC isolation. Controls are indicated when appropriate. The
isolation is divided into two phases: (1) processing of the sample from the operating room and establishment
of the primary culture, (2) establishment of MCBs and WCB and expansion of the culture (common to all MSC
culture regardless of tissue origin)
cell culture conditions. Flow cytometric analysis must demonstrate

expression of the surface markers CD105, CD73, CD29, and
CD90 and show minimal or null expression of CD45, CD34,
CD14 or CD11b, CD79, CD19 and HLA Class II proteins. MSCs
must demonstrate the ability to differentiate with proper culture
conditions to osteogenic, adipogenic, and chondrogenic lineages
(though chondrogenic differentiation is not usually attained due
to difficulty of the process). Here, we describe protocols for flow
cytometric surface phenotyping and osteogenic and adipogenic
in vitro differentiation.
3.3.1 Cell Surface 1. Collect approximately 2 × 105 cells from Passage 1 (to pheno-
Phenotyping type MCBs) and Passage 3 (to phenotype WCBs). It is recom-
mended that you type each batch of MSC cultures. Use the
following markers for phenotyping: CD105/CD31/CD166/
CD29/CD45RA/CD73.
2. Aliquot 20,000 cells into flow cytometry tubes, one for each
marker plus controls (see Note 7). Centrifuge at 400 × g for
5 min at 4 °C to pellet. Discard the supernatant.
3. Wash through three repeated cycles of centrifugation with PBS
staining buffer.
4. Block nonspecific antibody binding with 1 μL of appropriate
normal serum (usually mouse) for 10 min at 4 °C.
5. Add the labeled antibodies and isotype controls according to
manufacturer’s recommendations in 50 μL of PBS staining
buffer.
6. Incubate at 4 °C in the dark for 30 min.
7. Wash through three repeated cycles of centrifugation with PBS
staining buffer.
8. Resuspend in 400 μL of PBS staining buffer and measure by
conventional flow cytometry.
3.3.2 Adipogenic 1. Collect approximately 3 × 105 MCB cells from Passage 1

Differentiation (see Note 8).
2. Plate in triplicate in 6-well plates (105 cells/well). Add adi-
pocyte differentiation medium to 2 of them and leave 1 as
control.
3. After 3 weeks, add OIL-O-RED to the cultures to stain lipid
vacuoles characteristic of mature adipocytes.
4. Alternatively, differentiation may be quantified using flow
cytometry [14]. Fix cells with 0.5% paraformaldehyde for
15 min, then stain cells with 1 mg/mL Nile Red (Sigma; St
Louis, MO) for 30 min on ice. Measure using conventional
flow cytometry in the red channel.
3.3.3 Osteogenic 1. Collect approximately 3 × 105 cells from Passage 1 (MCB)

Differentiation (see Note 8).
2. Plate in triplicate in 6-well plates (105 cells/well). Add adipocyte
differentiation medium to 2 of them and leave 1 as control.
3. After 3 weeks, fix cells using 70% ethanol.
4. Stain with 2% alizarin red S for 10 min. Wash plates twice with
distilled water. Osteogenic differentiation should be readily
observed as red staining at the bottom of the wells [15].
5. For quantification of staining, add 10% (water solution) acetic
acid to the well, heat to 85 °C for 10 min, and then cool on ice
for 5 min.
6. Neutralize the acid with ammonium hydroxide.
7. Read absorbance at 405 nm in a spectrophotometer.
A typical surface phenotyping and differentiation control can
be observed in Fig. 2.
Fig. 2 Surface phenotype and differentiation controls in MSCs. (a) Micrograph representing a typical bone
marrow-derived MSC culture. (b) Surface phenotype of a bone marrow-derived MSC sample. (c) Comparison
of the differentiation capacities of MSCs from bone marrow, lipoaspirate, umbilical cord, and dental pulp into
adipogenic and osteogenic lineages. Fibroblasts are shown as controls
4 Notes
1. The samples and all reagents and equipment must be handled

aseptically (wear disposable gloves and lab coats, and work
within a “class II” biological safety cabinet). Avoid mechanical
stress (resuspend and pipette gently, minimize pellet time, and
avoid air bubbles).
2. Carefully load the sample on the top of the gradient material,
initially slowly to prevent excessive mixing with the gradient
material. Start from the sidewall and carefully work your way
up until the whole sample lies on the top of the gradient
material.
3. Carefully remove the tubes from the centrifuge while not dis-
turbing the layering. Identify the BMMC containing inter-
phase (cloudy, opaque) above the barrier between Ficoll
(colorless) and plasma (yellow). Part of the Ficoll and the pel-
let containing erythrocytes and most of the granulocytes are
located under the barrier.
4. We recommend performing three passages in total to ensure
availability of sufficient material for stocking WCBs, prevent-
ing cell culture adaptation associated with extensive passaging,
which may compromise the biological function of the cells.
5. It is critical to ensure the explants are firmly adhered to the
plastic while maintaining the viability of the tissue. Therefore,
fragments should remain without culture medium for enough
time to adhere to the plastic but not too long so the tissue
starts to die. Typically, an incubation of 1–2 h is sufficient. If
the fragments detach then cells will not culture from those
explants.
6. Although proper sterility conditions must be observed at all
times, it is expected that some cultures will still carry
microbiological contaminants, as the organ of origin (mouth)
presents a plethora of microbiological flora. It is important to
test hDPSC isolations as soon as possible for possible contami-
nants before committing to secondary culture growth.
7. We recommend the use of fluorochrome-conjugated primary
antibodies, with isotype controls selected according to the
availability of primary antibodies.
8. Differentiation controls aim to establish the potential of the
MSC cultures. Therefore, it is recommended to perform this
control as early as possible (i.e., after establishing the primary
culture). Slow cycling cells, such as bone marrow-derived
MSCs or umbilical cord-derived MSCs, may require higher cell
density. We recommend 5 × 105 cells/well.
References
1. Friedenstein AJ, Gorskaja JF, Kulagina NN 9. Bartholomew A, Sturgeon C, Siatskas M et al
(1976) Fibroblast precursors in normal and (2002) Mesenchymal stem cells suppress lym-
irradiated mouse hematopoietic organs. Exp phocyte proliferation in vitro and prolong skin
Hematol 4:267–274 graft survival in vivo. Exp Hematol 30:42–48
2. Haynesworth SE, Goshima J, Goldberg VM, 10. Banfi A, Muraglia A, Dozin B et al (2000)
Caplan AI (1992) Characterization of cells Proliferation kinetics and differentiation poten-
with osteogenic potential from human marrow. tial of ex vivo expanded human bone marrow
Bone 13:81–88 stromal cells: Implications for their use in cell
3. Zuk PA, Zhu M, Mizuno H et al (2001) therapy. Exp Hematol 28:707–715
Multilineage cells from human adipose tissue: 11. Dominici M, Le Blanc K, Mueller I et al (2006)
implications for cell-based therapies. Tissue Minimal criteria for defining multipotent mes-
Eng 7:211–228 enchymal stromal cells. The International
4. Lee OK, Kuo TK, Chen WM et al (2004) Society for Cellular Therapy position state-
Isolation of multipotent mesenchymal stem ment. Cytotherapy 8:315–317
cells from umbilical cord blood. Blood 103: 12. Wang XY, Lan Y, He WY et al (2008)
1669–1675 Identification of mesenchymal stem cells in
5. Miura M, Gronthos S, Zhao M et al (2003) aorta-gonad-mesonephros and yolk sac of
SHED: stem cells from human exfoliated human embryos. Blood 111:2436–2443
deciduous teeth. Proc Natl Acad Sci U S A 13. Wang Y, Yu J, Deng Z et al (2007) Odontogenic
100:5807–5812 capability: bone marrow stromal stem cells ver-
6. Sarugaser R, Lickorish D, Baksh D et al (2005) sus dental pulp stem cells. Biol Cell 99:465
Human umbilical cord perivascular (HUCPV) 14. Cárcamo-Orive I, Tejados N, Delgado J et al
cells: a source of mesenchymal progenitors. (2008) ERK2 protein regulates the prolifera-
Stem Cells 23:220–229 tion of human mesenchymal stem cells without
7. Zuk PA, Zhu M, Ashjian P et al (2002) Human affecting their mobilization and differentiation
adipose tissue is a source of multipotent stem potential. Exp Cell Res 314:1777–1788
cells. Mol Biol Cell 13:4279–4295 15. Carcamo-Orive I, Gaztelumendi A, Delgado
8. Rebelatto CK, Aguiar AM, Moretao MP et al J et al (2010) Regulation of human bone
(2008) Dissimilar differentiation of mesenchy- marrow stromal cell proliferation and differ-
mal stem cells from bone marrow, umbilical entiation capacity by glucocorticoid receptor
cord blood, and adipose tissue. Exp Biol Med and AP-1 crosstalk. J Bone Miner Res 25:
(Maywood) 233:901–913 2115–2125
Chapter 14
Cryobanking Mesenchymal Stem Cells

Andrés Pavón, Izaskun Beloqui, Juan M. Salcedo, and Angel G. Martin
Abstract
Cryopreservation and storage of culture-expanded mesenchymal stem cells (MSCs) is essential for a biobank
to maintain a collection of cell lines for research and clinical use. Optimization of cryopreservation protocols
and methods to minimize damage to cells during freezing and thawing is critical to ensure reliable avail-
ability of viable cells. Controlling the freezing rate and the use of appropriate cryoprotectant, as well as
stable storage temperature, can minimize the negative effects on cell viability. In this chapter, protocols for
cryopreserving MSCs are described.
Key words Cryopreservation, Cryoprotectant, DMSO, Viability, Control rate freezing, Mesenchymal
stem cells
1 Introduction
Cell-based research and therapy relies upon the availability of viable cells
fit for purpose. Human cells in particular require careful handling
and are in many instances irreplaceable. Therefore, it is vital that cells
are appropriately cryopreserved for storage of stocks over short- and
long-term. In the context of a cell biobank, optimal and quality
assured cell freezing and holding as characterized units is core to its
function for providing cells for research and/or clinical use. Once a
cell line is stable for consistent culture (see Chapter 13), then frozen
master cell stocks should be generated for limited access and use.
Working frozen cell stocks should also be prepared and replenished as
required. A cell line is considered extinguished if master cell stocks are
depleted. MSCs can be stored in a complete medium or animal serum
in the presence of a cryopreserving agent such as dimethylsulfoxide
(DMSO). DMSO is used as a cryoprotectant because of its high cell
permeability, however, exposing cells to DMSO at high concentra-
tions or for prolonged periods of time is damaging to the cells. A
concentration of 10% DMSO has been shown to be protective for
MSCs, preserving cell function in subsequent culture [1], although
5% DMSO combined with control slow-rate freezing also maintains
the differentiation potential of MSCs [2].
191
192 Andrés Pavón et al.
2 Materials
2.1 Reagents 1. DMEM: Dulbecco’s modified Eagle’s Medium-Low glucose

(Sigma; St. Louis, MO).
2. Dimethyl sulfoxide (DMSO; Sigma; St. Louis, MO).
3. Phosphate buffered saline (PBS; pH 7.2; Invitrogen;
Carlsbad, CA).
4. Fetal Bovine Serum (FBS).
5. Trypan Blue.
6. Trypsin-EDTA solution 0.25%, sterile-filtered, bioreagent,
suitable for cell culture: 2.5 g porcine trypsin and 0.2 g EDTA
per liter of Hank’s Balanced Salt Solution with phenol red
(Sigma; St. Louis, MO; Ref: T4049).
7. Freezing medium: To prepare 1 mL of Freezing Medium, add
900 μL FBS to 100 μL of DMSO (see Notes 1 and 2).
2.2 Consumables 1. 2 mL cryo tubes and screw cap. Internal thread Polypropylene
and Equipment (Nunc; Rochester, NY; ref.: 377267).
2. LN2 safe adhesive labels.
3. Sterile pipettes.
4. Sterile tubes with conical bottom 50 mL (Becton-Dickinson;
Franklin Lakes, NJ; ref.: 352098).
5. Control rate freezer or freezing container (see Note 3).
3 Methods
3.1 Cell Preparation Carry out all procedures at 4 °C. Cells should be frozen when they
are healthy and actively growing.
1. Clean biosafety cabinet (BSC) surfaces with 70% ethanol.
Sterilize all materials (pipettes, micropipettes, etc.) that are to
be used inside the BSC. Spray gloved hands and all bottles of
liquid with 70% ethanol.
2. Observe the cells under the microscope to confirm that they
are 80–90% confluent (see Note 4).
3. Carefully aspirate the culture medium using a vacuum-attached
pipette, taking care not to disturb the adherent cells.
4. Carefully wash the cells by adding 1× PBS (see Note 5). The
volume of PBS used should equal the original volume of
medium volume. Carefully remove the 1× PBS as in step 3.
5. Add tempered Trypsin-EDTA to detach the cells, using a suf-
ficient volume to cover the entire cellular surface and lightly
Cryobanking Mesenchymal Stem Cells 193
shake flask (see Note 6). Place the flask in the incubator for not
more than 5 min (see Note 6).
6. Stop Trypsin-EDTA action by adding temperate medium
containing DMEM + 10% FBS at a 1/3 dilution. Collect the
mixture into a labeled centrifuge tube. Using a microscope,
confirm that there are no or few cells left in the flask. Centrifuge
at 290–453 × g for 5–10 min at room temperature.
7. Remove the supernatant without disturbing the pellet.
Carefully resuspend the pellet with an appropriate volume (at
least 5 volumes of pellet size) of temperate medium containing
DMEM + 10% FBS.
8. Count the cells with a Neubauer chamber, diluting a small
aliquot of cells in trypan blue. Proceed with freezing cells if
viability is 90% or higher.
9. Centrifuge at 290–453 × g for 5–10 min at room
temperature.
10. During centrifugation, start to prepare labeled cryotubes.
Labels should include information such as cell line name, num-
ber of cells per tube, date of freezing, master or working cell
stock, etc. Place the empty cryovials in ice.
11. Remove the supernatant as in step 3.
12. Carefully resuspend the cells in a volume (for 1 mL per cryo
tube) of freezing medium containing 90% FBS and 10% DMSO
to achieve desired cell concentration (see Notes 7 and 8). All
subsequent steps should be done as quickly as possible.
13. Pipet 1 mL of the mixture in the cooled cryovials.
14. Close the cryovials and place them into the freezing container
(see Subheading 3.2) or control rate freezer (see Subheading 3.3).
3.2 Cryopreservation 1. If using a freezing container, store the freezing box at −80 °C
with a Freezing for at least 24 h prior to moving to long-term storage in a LN2
Container tank.
2. After freezing, test cell viability upon thawing in one vial of
each batch after 2 weeks in LN2 storage using trypan blue
viability staining. Viability should stay above 90% for optimal
results.
3.3 Cryopreservation A significant obstacle for cryopreserving MSCs arises from poor
by Control Rate survival upon freezing. Slow control rate freezing, either using a
Freezing homemade device or a sophisticated commercial freezing unit,
allows a controlled and reproducible means of cryopreserving
MSCs. As the water converts to ice, the cell dehydrates, changing
the concentration of salts and metabolites, and creating an osmotic
imbalance that may be detrimental to cell viability. Intracellular
ice-formation occurs during fast freezing and if the cell cannot

cope with the osmotic pressure it will die (refer to Chapter 5 for
further explanation of slow freezing and vitrification). To ensure
the highest survival level, it is critical to achieve a slow ice crystal
seed at −7 to −10 °C, with a freezing rate between 0.3 and 1.8 °C/
min. This is achieved through cooling an external portion of the
freezing tube for subsequent freezing of the cryopreservation
medium. In the presence of DMSO, the cell interior maintains its
liquid state. When the cryopreservation medium is frozen, water
exits the cell to equilibrate the salt concentration between the cell
exterior and inside the cell. Without an ice crystal seed, the cells
remain hydrated and the two fluid areas (internal and external)
freeze spontaneously al −20 °C. When the freezing process contin-
ues after the seed, the intracellular environment continues to dehy-
drate so that intracellular ice crystals do not form at −20 °C [3].
Automated control rate freezers allow precise control of the freez-
ing temperature over a given period of time. In addition, control
rate freezing can be scaled up and recorded both desirable features
for biobanking.
A control rate freezer usually consists of two parts:
• Magnetic stirrer and vortex breaker that provides unobstructed
use of a bath to ensure isothermal conditions.
• A fluid component (usually methanol) that is mechanically
refrigerated (see Note 9).
A protocol for control rate freezing MSCs that works best in
our hands is as follows:
1. Begin fast cooling from room temperature down to −10 °C at
0.5 °C/min.
2. Hold at −10 °C for 5 min. This step is critical to ensure the
formation of the ice crystal seed.
3. Cool down to −80 °C at 1 °C/min (target temperature).
4. Plunge into LN2 for subsequent storage (see Note 10).
Figure 1 depicts a typical temperature curve for control rate
freezing.
5. After freezing, test cell viability upon thawing in one vial of each
batch after 2 weeks in LN2 storage using trypan blue viability
staining. Viability should stay above 90% for optimal results.
4 Notes
1. The freezing medium should be prepared fresh for use and

mixed thoroughly.
2. DMSO facilitates entry of organic molecules into tissues. Always
handle hazardous materials using appropriate safety practices.
Cryobanking Mesenchymal Stem Cells 195
Fig. 1 Schematic of a typical freezing curve from a BioCool control rate freezer set from +10 °C to −80 °C. X
axis represents temperature (°C) while Y axis represents time (min). Note the hold step at −10 °C to allow for
ice crystal seeding
3. Freezing containers (such as the Thermo Scientific “Mr.

Frosty” -ref.: 5100-0001-) provide a simple-to-use system
designed to achieve a rate of cooling very close to −1 °C/min,
the optimal rate for cell preservation. Freeze cells in tubes from
1 to 5 mL depending on the size of container used. Store at
room temperature when not in use. Replace alcohol after every
fifth use.
4. Do not manipulate two samples or lines at the same time.
Clean and sterilize the hood between samples or lines.
5. Add 4–6 mL 1× PBS to a T25 flask, 13–18 mL to a T-75 flask,
or 20–25 mL to a T175 flask.
6. Add 1 mL trypsin to a T25 flask, 3 mL to a T-75 flask, and
5 mL to a T175 flask. It is important to observe the flask by
microscope during detachment procedure. All the cells have to
be detached and well disaggregated, without clumps. If the
cells are not detached after 5 min, tap the side of flask. Ensure
clumps of cells are dispersed.
7. The cell resuspension step is tricky and requires slow methodi-

cal mixing. Initially, the pipette or tip will be full of medium.
Release half of the volume and suck back up. Repeat three to
five times until no visible cell clumps can be seen. Then release
all liquid from the pipet tip. Suck up from the bottom and
release liquid at the top. Try to minimize air bubble formation
during mixing, as bubbles can destroy cells.
8. Freeze MSCs at a highest possible concentration (106 cells/vial
for a working cell stock or 0.5 × 106 cells/vial for a master cell
stock), making sure the cells are at least 90% viable at the time of
freezing. If the quantity of cells being frozen requires more than
15 vials, it is recommended that the cells be frozen in multiple
batches to reduce DMSO contact time. The time that cells are
in contact with DMSO should be minimized until frozen.
9. In order to have a consistent freezing process it is important to
change the methanol frequently, as it will become contami-
nated with water vapor.
10. Control rate freezers can be programmed to maintain a desired
temperature for prolonged periods of time once the target
temperature is achieved. It is recommended to set this param-
eter as “forever” (or equivalent), so the cells will be kept to
temperature until transferred to a LN2 tank or freezer; prevent-
ing unexpected thawing if this operation cannot be performed
on schedule.
References
1. Nicol A, Nieda M, Donaldson C et al (1996) human mesenchymal stromal cells isolated for
Cryopreserved human bone marrow stroma is clinical use. Cytotherapy 9:328–337
fully functional in vitro. Br J Haematol 94: 3. Ware CB, Baran SW (2007) A controlled-
258–265 cooling protocol for cryopreservation of human
2. Haack-Sorensen M, Bindslev L, Mortensen S and non-human primate embryonic stem cells.
et al (2007) The influence of freezing and Methods Mol Biol 407:43–49
storage on the characteristics and functions of
Part IV
Protocols for Human Neural Stem Cell Banking

Chapter 15
Culturing and Cryobanking Human Neural Stem Cells

Jeremy M. Crook and Eva Tomaskovic-Crook
Abstract
The discovery and study of human neural stem cells has advanced our understanding of human neurogenesis,
and the development of novel therapeutics based on neural cell replacement. Here, we describe methods
to culture and cryopreserve human neural stem cells (hNSCs) for expansion and banking. Importantly, the
protocols ensure that the multipotency of hNSCs is preserved to enable differentiation to neurons and
supporting neuroglia.
Key words Human neural stem cells, Culture, Expansion, Passaging, Freezing, Cryopreservation
1 Introduction
Human neural stem cells (hNSCs) hold great promise for both
medical application and as a research tool for addressing fundamen-
tal questions in development and disease [1]. While sourcing tissues
for primary hNSC isolation and line derivation can be difficult,
off-the-shelf ready-to-use hNSCs are available from a number of
suppliers for research and clinical use. Here, we describe in detail
methods to culture hNSCs for expansion, and freezing for cell
banking. The protocols are derived from previously published work
that can be referred to for additional information about working
with hNSCs (i.e., to characterize hNSCs and their progeny) beyond
the scope of the present chapter [2]. Similar to other stem cell types
described in this volume, a desired level of quality assurance can be
applied for cell culture, freezing, and storage. In any case, standard-
ized and reliable protocols are necessary for basic science through
to applied research and clinical product development. This chapter
details methods that are suitable for routine application, high-quality
research and adaptable for clinical-compliance.
199
200 Jeremy M. Crook and Eva Tomaskovic-Crook
2 Materials
2.1 hNSC Culture 1. hNSCs (eg., Millipore: SCC007).

and Passaging 2. hNSC proliferation medium: NeuroCult® NS-A Proliferation
Kit, human (STEMCELL Technologies, Cat. no. 05751) (see
Note 1).
3. Penicillin Streptomycin (10,000 U/mL, Life Technologies,
Cat. no. 15140122).
4. EGF (Cat. no. AF-100-15) and basic-FGF (Cat. no. AF-100-
18B; Peprotech, Lonza).
5. Tris–HCl (Sigma, Cat. no. T5941).
6. Human serum albumin (Sigma, Cat. no. A9511).
7. TrypLE™ Select (Gibco, Life Technologies, Cat. no.
12563-029).
8. Heparin (Sigma, Cat. no. H3149).
9. DMEM/F12 (Gibco, Life Technologies, Cat. no. 11330-057).
10. Phosphate buffered saline (Sigma, Cat. no. P5368).
11. Ultra-low attachment 6-well plates (Corning Costar®, Cat. no.
CLS3471, Sigma).
13. CO2 incubator.
14. Benchtop centrifuge.
15. Pipetman and tips.
16. Pipet-Aid (motorized pipette) and serological pipettes.
18. Class 2 Biological Safety Cabinet (biosafety cabinet).
19. Sterilized Schott glass bottle, 100 mL.
2.2 hNSC 1. hNSC proliferation medium: NeuroCult® NS-A Proliferation

Cryopreservation Kit, human; STEMCELL Technologies, Cat. no. 05751)
and Thawing (see Note 1).
2. TrypLE™ Select (Gibco, Life Technologies, Cat. no.
12563-029).
3. DMEM/F-12 (Gibco. Life Technologies, Cat. no. 11330-057).
4. Isopropyl Alcohol (Chem-Supply, Cat. no. 323-4).
5. 95% Ethanol.
6. CryoStor® CS10 (BioLife Solutions, Cat. no. 99-610-DV;
STEMCELL Technologies).
Culturing and Cryobanking Human Neural Stem Cells 201
9. 1.5 mL Cryovials (Nunc, Cat. no. 5000-1020).

10. Ultra-low attachment 6-well plates (Corning Costar®, Cat. no.
CLS3471; Sigma).
11. −80 °C freezer.
12. Benchtop centrifuge.
14. CO2 incubator.
15. Pipet-Aid (motorized pipette).
16. Ice bucket.
17. LN2 storage tank.
18. Isopropanol freezing units (Nalgene®, Cat. no. 5100-0001,
Cryo 1 °C “Mr. Frosty” Freezing Container, ThermoFisher
Scientific) or equivalent non-isopropanol freezing units
(CoolCell: Corning, Cat. no. 432001).
19. Cryovial racks.
20. Metal forceps.
21. Sterilized Schott glass bottle, 100 mL.
3 Methods
Reagent preparation and cell culture work should be performed in a

biosafety cabinet unless otherwise specified. All media and reagents
should remain sterile. Incubations and culturing should be performed
in a 37 °C incubator with a humidified atmosphere of 5% CO2 in air.
Centrifugation steps are performed at room temperature (RT).
3.1 hNSC Culture hNSC can be cultured as neurosphere suspension cultures or as

and Passaging adherent monolayer cultures. The method outlined below details
neurosphere suspension culture.
1. Prepare hNSC culture medium (e.g., ~500 mL):
(a) Neurocult media: 450 mL.
(b) Neurocult supplement: 50 mL.
(c) EGF (200 μg/mL stock): 50 μL (for 20 ng/mL working
solution). To prepare 200 μg/mL EGF stock solution, dis-
solve 500 μg vial of EGF in 2.5 mL filter sterilized 5 mM
Tris–HCl containing 0.5% human serum albumin, pH 7.6.
Store 50 μL aliquots of 200 μg/mL EGF stock solution at
−80 °C. Do not freeze/thaw each vial more than twice.
Add 50 μL to 500 mL Complete hNSC Proliferation
Medium. If preparing 100 mL Complete hNSC
Proliferation Medium, add 10 μL to 100 mL Complete
hNSC Proliferation Medium. Aliquot unused portion to
4 × 10 μL aliquots of 200 μg/mL EGF stock solution at

−80 °C for later use.
(d) bFGF (200 μg/mL stock): 50 μL (to get 20 ng/mL work-
ing solution). To prepare 200 μg/mL bFGF stock solu-
tion, dissolve 500 μg vial of bFGF in 2.5 mL filter sterilized
5 mM Tris–HCl containing 0.5% human serum albumin,
pH 7.6. Store 50 μL aliquots of 200 μg/mL bFGF stock
solution at −80 °C. Do not freeze/thaw each vial more
than twice. Add 50 μL to 500 mL Complete hNSC
Proliferation Medium. If preparing 100 mL Complete
hNSC Proliferation Medium, add 10 μL to 100 mL
Complete hNSC Proliferation Medium. Aliquot unused
portion to 4 × 10 μL aliquots of 200 μg/mL bFGF stock
solution at −80 °C for later use.
(e) Heparin (2 mg/mL stock): 500 μL (to get 2 μg/mL
working solution) (see Note 2). Store prepared 500 μL ali-
quots of 2 mg/mL heparin stock solution in PBS at
−20 °C. Add 500 μL to 500 mL Complete hNSC
Proliferation Medium. If preparing 100 mL Complete
hNSC Proliferation Medium, add 100 μL to 100 mL
Complete hNSC Proliferation Medium. Aliquot unused
portion to 4 × 100 μL aliquots of 2 mg/mL Heparin stock
solution at −20 °C for later use.
(f) To prepare hNSC Proliferation Medium with cytokines for
expansion of hNSCs requires addition of heparin (2 μg/
mL; Sigma), epidermal growth factor (EGF, 20 ng/mL;
Peprotech), and basic fibroblast growth factor (bFGF,
20 ng/mL; Peprotech).
(g) To ensure stability of components, prepare sufficient work-
ing volume of Complete hNSC Proliferation Medium
(with additives) for 2–3 weeks expansion of hNSCs at any
one time. To prepare a 100 mL volume of Complete hNSC
Proliferation Medium (without additives), thaw 50 mL
bottle of NeuroCult NS-A Proliferation Supplement at RT
for 3–4 h until just thawed. Mix well. Aliquot NeuroCult
NS-A Proliferation Supplement into 10 mL volumes in
4 × 15 mL tubes for storage at −20 °C until required. Add
remaining 10 mL contents to 90 mL NeuroCult NS-A
Basal Medium in sterile 100 mL bottle. Mix well. Complete
hNSC Proliferation Medium can be stored without cyto-
kines for up to 4 weeks. To prepare Complete hNSC
Proliferation Medium with cytokines requires addition of
heparin (2 μg/mL; Sigma), epidermal growth factor
(EGF, 20 ng/mL; Peprotech), and basic fibroblast growth
factor (bFGF, 20 ng/mL; Peprotech). Complete hNSC
Proliferation Medium (with additives; ADDS) can be
stored at 4 °C for 2–3 weeks.
2. Seed hNSCs at a density of 2–3 × 106 cells in 2 mL Complete

hNSC Proliferation Medium (with ADDS) per well of an ultra-
low attachment 6-well plate. Label plates with cell line, passage
number, date, and initials.
3. Return cultures to 37 °C incubator. Agitate the plates daily to
help prevent neurospheres from fusing together.
4. Monitor cultures daily and inspect morphology (semi-
transparent, microspikes on outer surface of neurosphere),
neurosphere size (growth to less than 100–150 μm in diameter)
and acidity of media (change media before reaching orange/
yellow color).
5. Feed cultures every 3–4 days with a half media change. To
replenish media, collect medium containing neurospheres into
a 15 mL tube and centrifuge at 190 × g for 3 min. Aspirate the
half the volume of the medium. Replace with equal volume of
pre-warmed Complete hNSC Proliferation Medium (with
ADDS). Gently agitate the pellet with a 5 mL serological pipette
to separate the cells. Return medium containing neurospheres
to same wells.
6. Passage cells approximately every 7–10 days so as not to allow
neurospheres to exceed 100–150 μm diameter. Collect medium
containing neurospheres into a 15 mL tube and centrifuge at
190 × g for 3 min.
7. Aspirate the medium without disturbing the pellet (see Note 3).
8. Add 1 mL pre-warmed TrypLE to the tube and gently mix
tube contents by flicking tube.
9. Place the tube in a water bath at 37 °C for 3 min.
10. Gently agitate the pellet with a 1 mL pipette to separate the
cells (see Note 4). Add 5 mL Complete hNSC Proliferation
Medium (without ADDS) (or aspirate from step 4, if available)
to stop the digestion. Centrifuge at 190 × g for 3 min.
11. Aspirate the supernatant and resuspend the cells with fresh
pre-warmed Complete hNSC Proliferation Medium (with
ADDS). Count number of viable cells with a hemacytometer
using Trypan Blue exclusion assay.
12. Seed the cells by repeating step 2.
13. Label plates with cell line, passage number, date, and initials.
Return cultures to 37 °C incubator.
3.2 hNSC Neurospheres cultures that have been grown for at least 3–4 days
Cryopreservation and do not exceed 100–150 μm diameter can been frozen down
as whole neurospheres, or single-cell populations. The method
outlined below details the cryopreservation of single-cell
populations.
1. Determine number of vials to be cryopreserved (1 cryovial for

each 6-well plate of confluent cells), and label vials appropri-
ately. Minimally including cell line name, passage number, and
date frozen (see Note 5).
2. Collect medium containing neurospheres from wells of each
6-well culture plate and combine contents into separate 15 mL
tubes (one plate of cells per tube).
4. Aspirate the medium without disturbing the pellet.
5. Add 1 mL pre-warmed TrypLE to each tube and gently mix
tube contents by flicking tube.
6. Place the tube(s) in a water bath at 37 °C for 3 min.
7. Gently agitate the pellet(s) with a 1 mL pipette to separate the
cells (see Note 4). Add 5 mL Complete hNSC Proliferation
Medium (without ADDS) (or aspirate from step 4, if avail-
able) to stop the digestion. Centrifuge at 190 × g for 3 min.
8. Aspirate the supernatant and gently resuspend the cells with
1 mL cold (stored at 4 °C, keep on ice) CryoStor CS10 solu-
tion (cryopreservation media).
9. At this point cells in cryopreservation media prepared from
different plates can be pooled into an appropriate sized tube
(e.g., 50 mL centrifuge tube).
10. Using an appropriately sized serological pipette, transfer 1 mL
volume of cells in cryopreservation medium to a prepared cryovial,
being careful not to contact the pipette tip with the cryovial.
Repeat until the pooled cell suspension is exhausted.
11. Cap each cryovial securely, quickly place them into the room
temperature Mr. Frosty or CoolCell container, and transfer the
container into the −80 °C freezer overnight.
12. 24 h later, remove the frozen cryovials from containers in the
−80 °C freezer, and place into LN2 storage.
3.3 Thawing hNSCs It is important that safety glasses be worn during this procedure, as
while it is a rare occurrence, sealed vials can explode during the
thaw process.
1. Pre-warm 1 mL Complete hNSC Proliferation Medium
(with ADDS) per well of an ultra-low attachment 6-well plate.
In general, a single frozen vial can be thawed into 3 wells of a
6-well plate. Add 5 mL pre-warmed Complete hNSC
Proliferation Medium (without ADDS) to a 15 mL tube for
each cryovial to be thawed.
2. Retrieve the vial to be thawed from LN2 storage and transport
to the lab in a dewar filled with LN2 or dry ice.
3. Confirm the cap is tightly closed, and using metal forceps,

immerse the vial into a 37 °C water bath without submerging
the cap. Swirl the vial gently in the water.
4. When only a small ice crystal remains, remove the vial from the
water bath.
5. Confirm again that the vial cap is tightened, and immerse the
vial into 95% ethanol.
6. In the biosafety cabinet, open the vial being careful not to
touch the rim of the vial.
7. Using a sterile 1 mL pipette, remove the cell suspension and
gently transfer into a sterile 15 mL conical tube contained pre-
warmed hNSC media. Slowly add contents of cyrovial dropwise
to the hNSC media, gently moving the tube back and forth to
mix. It is critical to add the cells slowly to avoid osmotic chock
to the cells.
9. Aspirate the supernatant, being careful not to disturb the cell
pellet (see Note 6).
10. Resuspend the cell pellet in Complete hNSC Proliferation
Medium (with ADDS) per cryovial so that there will be a final
volume of 2 mL per well total in the prepared culture plate
after adding the cells (i.e., if plates were prepared with 1 mL of
media per well, and the vial will be thawed into 3 wells, resus-
pend in 3 mL medium, ultimately adding 1 mL of suspension
to the 1 mL in the plate for a total of 2 mL).
11. Slowly add the cell suspension dropwise to the prepared plate
for recovery and expansion.
12. Place plate gently into the incubator. Distribute the cells/neuro-
spheres by gently shaking the plate front to back, rest a few
seconds, then side to side, and close the incubator door gently.
13. Passage cells after 5–7 days of recovery post-thawing.
4 Notes
1. NeuroCult® NS-A Proliferation Kit, human; STEMCELL

Technologies, 05751) consists of two components (NeuroCult
NS-A Basal Medium (Stem Cell STEMCELL Technologies,
Human; 05750; 450 mL) and NeuroCult NS-A Proliferation
Supplement (STEMCELL Technologies, Human; 05753;
50 mL)) which are mixed to prepare Complete hNSC
Proliferation Medium. Complete hNSC Proliferation Medium
can be stored without cytokines for up to 4 weeks.
2. Heparin increases the affinity of bFGF for its receptors.
3. The aspirated medium can be collected for use in step 7 to

inactivate TrypLE.
4. Do not flux the cells by pipetting more than seven times.
5. The labeled passage number should be increased by one com-
pared to the harvested culture to be frozen. Therefore, the culture
vessel to be labeled at thaw will match the vial being thawed.
6. Vacuum aspiration should be used with extreme caution, as
there is a significant risk of aspirating the cell pellet. If the pel-
let is smaller than anticipated, or if the cells are particularly
valuable or rare, use a sterile serological pipette to remove the
supernatant.
Acknowledgment
The authors wish to acknowledge funding from the Australian

Research Council (ARC) Centre of Excellence Scheme
(CE140100012).
References
1. Crook JM, Kobayashi NR (2008) Human stem 2. Kobayashi N, Sui L, Tan PSL et al (2010)
cells for modeling neurological disorders: accel- Modelling disrupted-inschizophrenia 1 loss of
erating the drug discovery pipeline. J Cell function in human neural progenitor cells:
Biochem 105:1361–1366 tools for molecular studies of human neurode-
velopment and neuropsychiatric disorders. Mol
Psychiatry 15:672–675
INDEX
A Clinical application ................4, 8, 17–20, 23, 51, 58, 59, 68, 69,
79, 85, 87, 89, 93, 99–101, 103–106, 108, 110, 115
Acquisition ...................................................................17–23 Clinical-grade .........................................4, 7, 12, 13, 68, 101,
Adaptation.....................................15, 92, 139–150, 186, 189 102, 104, 107, 116, 127, 140
Adipogenic ....................................................... 183, 187, 188 Clinically-compliant ......................................... 32, 51, 67–69
Adipose ............................................................ 177, 178, 183 Clinical translation ........................................... 104, 107, 108
Adult stem cells ................................................ 101, 102, 107 Clinical trials ............................................................ 100, 178
Allogeneic......................................................... 100, 177, 178 Clonal ....................................................... 135, 169–170, 172
Anonymized .................................................................19, 82 Code ................................................31, 82, 83, 103–105, 108
Apoptosis.......................................................... 53, 62, 64–66 Collagenase .............................. 140, 152, 157–159, 161, 163,
Australia ............................................................. 14, 102–104 164, 179, 183, 185
Autologous ......................................................... 20, 101, 177 Colonies ............................... 47, 56–59, 63, 67, 92, 134, 136,
137, 144, 149, 158, 159, 161, 163, 170, 172
B
Colony ........................................ 21, 56, 57, 59, 62, 133, 134,
Banking ...........3–9, 11–14, 17–23, 43, 58, 66, 69, 79, 84–87, 136, 149, 155, 156, 161, 163, 172
89, 93, 101, 104, 109, 110, 141, 151, 156, 161, 199 Comparative genome hybridization (CGH) ...........15, 94, 141
Batch ........................................... 61, 131, 187, 193, 194, 196 Conflicts of interest ..........................................................108
Beloqui, I. ......................................29, 36, 177–189, 191–196 Consent ........................................ 7, 9, 13, 14, 18–20, 23, 30,
Best practice ............................... 4, 15, 18, 20, 21, 23, 67, 79, 81–84, 103, 105, 106, 109, 110, 177
81, 103–106, 108, 109 Contamination .......................................3, 15, 20, 42, 57–59,
Bio-specimen.................................................... 17, 23, 30, 36 66–68, 80, 82, 85, 86, 91, 94–96, 145, 148, 154, 161,
Bisection ................................................... 116, 123–125, 128 172, 183, 185, 196
Blastocyst.................................................4, 58, 115–127, 154 Controlled-rate freezing (CRF) ............................ 38, 61–62,
Blomberg, P. .................................................................11–14 120, 152–154, 161–163
Bone marrow ..........................................45, 46, 48, 177, 178, Cooling rate................................................ 44–52, 55, 57–62
180–181, 184, 186, 188, 189 Crook, J.M................. 115, 121, 122, 125, 151–164, 199–206
Brehm, J.L. ...............................................................139–150 Cryopreservation..................................... 38, 41–68, 80, 86, 87,
Bruce, K.................................................51, 79, 85, 87, 89, 93 91, 92, 95, 119, 127, 135, 151, 155, 157, 159, 160, 163,
193–194, 200–201, 203–204
C Cryoprotectant .................. 46, 51–57, 60, 63–64, 68, 69, 191
Campbell, J.D.M. ........................................79, 85, 87, 89, 93 Cryoprotection ...................................................................64
Carlson-Stevermer, J.................................................165–173 Cryovial ............................................31, 57–59, 66, 153–156,
Cell banking ................................... 43, 58, 59, 84–86, 88, 92, 159, 160, 193, 201, 204, 205
110, 141, 161, 199 Culture ........................................... 3, 12, 18, 30, 42, 80, 101,
Cell identity ...............................................18, 20, 42, 81, 115 115, 131, 139, 152, 166, 178, 191, 199
Cell line history ..............................................................7, 86
D
Cell potency ........................................................... 81, 90–91
Cell therapy .......................................9, 42, 81, 101, 104, 177 Defined.......................................................15, 35, 45, 49, 80,
Chain of custody .................................................... 18, 31, 32 84–86, 128, 129, 131–137, 140, 151, 152
Chalmers, D. ......................................................................99 Dental pulp............................................... 178, 184–186, 188
Characterization .......................................8, 9, 13, 15, 20, 23, Depositors ................................................................104–107
79–97, 99, 104, 108, 115, 133 de Sousa, P. ..................................................79, 85, 87, 89, 93
Chemically defined............................................. 15, 131–137 Derivation....................................... 19, 20, 41, 68, 69, 80, 94,
Chen, G....................................................................131–137 99, 101–102, 115–129, 131–137, 140
DOI 10.1007/978-1-4939-6921-0, © Springer Science+Business Media LLC 2017
207
STEM CELL BANKING: CONCEPTS AND PROTOCOLS
208 Index
Differentiation .................................... 8, 9, 42, 53, 56, 57, 60, Freezing .................................................12, 13, 38, 42–56, 61
62, 65, 66, 90, 92, 107, 144, 145, 147, 149, 157, 172, , 63, 65, 119, 120, 151, 152, 154–163, 173, 191–196,
177–179, 184, 186–189, 191 199, 201
Dimethyl sulfoxide (DMSO) .................... 51–54, 56, 59–64,
66–68, 152, 155, 157, 159, 163, 191–194, 196 G
Dispase .......................135, 140, 146, 157, 158, 163, 179, 185 G-banding .............................................................. 87, 92–94
Dissociation ........................... 56, 65, 133, 135, 169, 171, 173 Genetic identity ............................................................86–88
Distribute ...............20, 88, 105, 106, 144, 145, 149, 161, 205 Genetic stability ............................................. 16, 92–94, 141
Documentation.................... 12, 22, 23, 82, 86, 100, 108, 109 Genetically-modified............................................ 21, 66, 107
Donor ..........................4, 9, 12, 14–16, 19, 20, 23, 30, 31, 37, Genome editing ........................................................165–173
81–83, 86–88, 94, 95, 100, 110, 165–167, 171, 177 Genotyping ........................................................ 5, 21, 30, 87
Dulbecco’s Modified Eagle Medium (DMEM) ...............117, Germ layers .................................................4, 87, 90, 91, 141
118, 122, 132–134, 141–143, 146, 148, 149, 152, 155, Giemsa staining ..................................................................93
157, 158, 163, 167, 168, 178, 179, 183, 185, 192, Good laboratory practice (GLP) ...................... 12, 15, 18, 30
193, 200 Good manufacturing practice (GMP) ................... 12, 13, 15,
18, 30, 51, 67–69, 132
E
Governance ........................................................ 7, 18–20, 99
Efficiency...................................... 8, 131, 132, 134–136, 139, Growth profiling .................................................... 88, 91–92
150, 159, 166, 168–171, 177 Guardianship ............................................................105–106
Electroporation.........................................................166–172
Embryo..................................... 5, 8, 9, 20, 47, 56–58, 67, 80, H
95, 99, 101–103, 115–129, 151
Harmonization ........................................................... 23, 109
Embryoid body ....................................................... 64, 87, 90
Healy, L. .............................................................................17
Embryonic stem cells (ESCs)
Hermetically sealed ..........................................................161
colony picking...............................................................56
Hovatta, O. ...................................................................11–14
culture ......................................................... 123, 158, 159
Human leukocyte antigens (HLAs) ..................... 87, 88, 187
lines .................................... 4, 5, 8, 20, 41, 58–60, 68, 89,
Hunt, C.J. .....................................................................41–68
100, 104, 107, 108, 115, 116, 119, 126, 128, 172
medium............................................... 117, 118, 123–126 I
thawing ............................................42, 62, 119, 126, 151
transfection ................................................. 166, 167, 169 Immunogenicity ...............................................................178
Embryo transfer ....................................... 122, 124, 126, 128 Immunosurgery ........................................ 117–118, 123–125
Engineering ......................................................................165 In vitro fertilization (IVF) ........................ 116–120, 122, 124
Equilibrium ..................................... 43, 44, 46, 48, 49, 53, 55 Induced pluripotent stem cell (iPSCs)........................ 4, 5, 8, 9,
Ethical conduct ................................................ 103, 105, 108 11–14, 17, 18, 20, 21, 42, 81, 84, 91, 93, 101, 131–137,
Ethics ................................................7, 9, 13, 14, 19, 99–110 151, 172
Ethylenediaminetetraacetic acid (EDTA) ............... 133–137, Information flow ..........................................................30–31
140, 147, 157, 163, 179, 181, 182, 184, 185, 192, 193 Inner cell mass (ICM) ................................58, 115, 116, 118,
European Union (EU) ................................................ 15, 103 120, 122–125, 128, 129
Expansion ...............................................9, 15, 21, 56, 68, 80, Internal identification .........................................................37
84, 85, 90, 91, 107, 115, 135, 139–150, 156, 161, 163, International Society for Biological and Environmental
177–189, 199, 202, 205 Repositories (ISBER) ...........................................106
International Society for Stem Cell Research
F (ISSCR) .............................12, 14, 103, 104, 107, 108
International Standards Organization (ISO).......... 12, 13, 30
Feeder cells. See Human foreskin fibroblast; Mouse
International Stem Cell Banking Initiative
embryonic fibroblasts
(ISCBI).......................... 4, 12, 20, 100, 103, 104, 109
Feeder-free culture.................................................... 144, 150
International Stem Cell Forum (ISCF).............9, 20, 100, 103
Ferrin, I.....................................................................177–189
Intracellular ice ............................44, 46–50, 55, 62, 193–194
Fibroblasts ................................ 20, 21, 47, 68, 117, 118, 123,
Irradiated .................................................................. 117, 118
131, 133–136, 157, 183, 188, 202
Firpo, M.T. ............................................... 115, 121, 122, 125
J
Flow cytometry............ 15, 87–89, 91, 92, 133, 168, 170, 187
Framework ................................. 7, 12, 14, 17, 18, 20, 21, 36, Japan ........................................................................... 61, 102
68, 80, 100, 103–105 Jurisdictions ...................................................... 100, 103, 109
Index
209
K Osmotic equilibrium ....................................................43, 44

Osteogenic ................................................ 179, 183, 187–189
Kallur, T. .......................................................................11–14 Outgrowth ................... 80, 115, 116, 119, 125–126, 128, 129
Karyotype ........................................15, 92–94, 140, 141, 149
Kravets, L. ................................................ 115, 121, 122, 125 P
L Passaging .......................... 65, 85, 91, 92, 119, 125–126, 133,
136, 140, 144, 145, 147, 149, 150, 186, 189, 200–203
Labarga, A. ...................................................................29, 36 Patient-specific ................................................. 100, 101, 131
Laboratory information managing systems Pavon, A. ..................................................................191–196
(LIMS) .............................................................31–39 Performance metrics .....................................................37, 38
Laboratory workflows.........................................................37 Peura, T..................................................... 115, 121, 122, 125
Legal....................................................13, 14, 17, 31, 81, 105 Phenotype ................................. 15, 42, 87–92, 166, 187, 188
Legislation ..................................... 5, 14, 18, 19, 21, 102, 103 Pluripotent .......................4, 11, 17, 42, 79, 99, 115, 131, 140
Lipoaspirate ...................................................... 183, 186, 188 Pluripotent stem cells.................... 4–5, 7–8, 20–21, 42, 47, 48,
LN2 ............................................................... 38, 57, 58, 61, 62, 59–69, 79, 81, 88, 91, 99, 101, 104, 131–137, 139–173
66–68, 116, 120–122, 127, 143, 152–157, 160, 162, Polymerase chain reaction (PCR) .......................... 86, 87, 89,
170, 192–194, 196, 201, 204 91, 96, 169, 172, 173
Ludwig, T. ................................................................151–164 Pre-master cell bank .....................................................84, 85
Primary cultures........................................ 146, 180–186, 189
M Prions ...........................................................................83, 97
Management ..............5, 7, 11–13, 18, 21, 23, 29–39, 80, 105 Protocol ................................ 7, 8, 13, 42, 53, 55, 56, 59, 60, 62,
Martin, A.G. .................................29, 36, 177–189, 191–196 63, 65, 66, 68, 69, 86, 90, 116, 119, 132, 136, 140, 141,
Master cell bank (MCB) ..................................15, 84, 85, 92, 143, 147, 151, 157, 163, 167–171, 187, 194, 199
181, 183–185, 187, 188 Purity...................................................................... 13, 81, 91
Material Deposit and Distribution Agreement (MDDA) 106
Material transfer agreement (MTA) ............. 22, 23, 106, 110
Q
Matrigel ...................................... 63, 134, 136, 137, 140–144, Quality assurance (QA) ................................7, 11–16, 79, 85,
146–148, 157, 163, 167 87, 89, 93, 105, 107–108, 110, 161, 199
Media ............................................... 8, 15, 67, 68, 86, 90, 92, Quality control (QC)................................4, 8, 12, 15, 18–21,
94, 116, 123, 126, 127, 132–135, 140, 142, 155–157, 32, 37, 38, 42, 80, 83, 85, 100, 102, 109, 110
160–163, 166–170, 172, 173, 201, 203–205 Quality standards.................................................... 7, 29, 104
Medium ..........53, 96, 120, 131, 140, 152, 168, 179, 191, 200
Mesenchymal stem cells (MSCs) .....................48, 56, 64, 66, R
100, 101, 177–189, 191–196
Rao, M......................................................................131–137
Microbiological tests .............................................. 81, 94–97
Rathjen, J. ...........................................................................99
Mouse embryonic fibroblasts
Rathjen, P. ..........................................................................99
medium.........................................................................57
Reagents ..................7, 15, 68, 86, 95–97, 116, 122, 131, 140,
passaging ......................................................................56
142, 152, 157, 166, 167, 172, 178–179, 189, 192, 201
preparation....................................................................57
Regenerative medicine.......................6, 68, 99, 100, 139, 177
mTeSR1 ............................ 140–147, 149, 163, 166–168, 170
Regulations ................................ 19, 22, 81, 99, 102–110, 180
Mycoplasma ..................................................3, 20, 86, 87, 96
Report .................................... 4, 8, 31, 32, 37, 38, 41, 46–48,
56, 58–66, 68, 80, 105, 107, 116, 154, 184
N
Reprogramming ........................................... 20, 91, 131–136
National Institute for Biological Standards and Control Requestors ................................................................104–106
(NIBSC) ...................................................................5 Research ........................................... 3, 11, 17, 29, 42, 79, 99,
Neural stem cells ......................................................199–206 115, 131, 139, 151, 177, 191, 199
Nicol, D. .............................................................................99 Rewarming rate ..................................................................58
Nucleation ...................................... 43, 44, 46, 47, 49–51, 53, Rho-associated kinase (ROCK) inhibitors .................. 65–66,
54, 56, 61–63, 69, 162 134, 136, 137, 146, 167–170, 172
O S
Off-the shelf ............................................................. 100, 199 Safety .................................4, 7, 12, 15, 16, 18, 21, 22, 32, 42,
Oocytes....................................................................... 45, 101 79, 85, 87, 89, 93, 101, 103, 107–108, 110, 127, 142,
Operators.........................................57, 92, 93, 104, 128, 129 148, 154, 160, 161, 179, 180, 189, 194, 200, 204
210 Index
Saha, K. ....................................................................165–173 Transgene ................................................................. 166, 169

Salcedo, J.M..............................................................177–189 Transgenesis .....................................................................166
Sample Transportation .......................................... 22, 57, 66–69, 184
cession ..........................................................................37 Treatment ....................................... 8, 15, 31, 47, 82, 83, 100,
handling........................................................................37 101, 123, 140, 183, 184
movement .....................................................................31 Trigueros, C. .............................................................177–189
processing ..................................................... 30, 180–185 Trophectoderm ..................................115, 116, 120, 123, 125
procurement..................................................................30 Tryggvason, K...............................................................11–14
shipping ........................................................................31 Trypsin ......................................... 92, 96, 135, 136, 163, 179,
storage ..........................................................................31 181, 182, 184, 185, 192, 193, 195
Sendai virus ..............................................................131–136
Serum-free........................................................ 124, 125, 135 U
Short tandem repeats (STRs) ............................. 9, 20, 86, 87 UK Stem Cell Bank.............................4, 6, 68, 102, 105–108
Single nucleotide polymorphisms (SNPs) ................... 15, 87, Umbilical cord ................................................. 101, 177, 178,
88, 94, 166 181–183, 186, 188, 189
Slow-rate freezing ............................................................191 Undifferentiated ..................................56, 128, 140, 147, 149
Source .......................... 3, 8, 17, 18, 20, 21, 58, 61, 68, 83, 86, United Kingdom (UK) ...................................4–6, 19, 36, 59,
94, 96, 97, 99–101, 103, 107, 116, 127, 131, 163, 167 61, 68, 83, 102, 103, 105–108
Spain ................................................................ 6, 31, 36, 102 United States of America (USA)............................. 6, 14, 36,
Spectral karyotyping (SKY ) ................................... 87, 93, 94 102, 127, 141, 142, 147
Stacey, G.......................................................................3–4, 6
Standardization ........................... 23, 79, 80, 93, 99, 106, 110 V
Stem cell banking ...................... 3, 6, 11–14, 17–22, 104, 109
Vectors ................................................................ 91, 167, 171
Stenfelt, S. ....................................................................11–14
Viability ......................................................37, 38, 42, 45, 80,
Storage ...........................................13, 14, 18–20, 23, 29–31,
81, 87, 88, 91–92, 128, 140, 151, 154, 161, 172, 189,
33, 38, 42, 50, 57, 58, 66–69, 84, 86, 101, 104, 106,
193, 194
107, 109, 119, 143, 147, 148, 153, 154, 156, 157, 160,
Viral testing ..................................................................86, 87
162, 191, 193, 194, 199, 201, 202, 204
Viruses ............................................................ 20, 22, 96, 133
Straw ..........57–59, 61, 66, 120, 121, 127, 152–156, 161–163
Vitrification ......................................................... 41–68, 120,
T 152–156, 194
Teratoma ....................................... 8, 15, 59, 87, 90, 107, 141 W

Thawing
Warming rate.......................................................... 49, 55, 56
cells ..............................................42, 49, 50, 62, 156, 162
Working cell bank (WCB) ...............................42, 67, 84, 85,
embryos ...................................... 116–117, 119–122, 127
92, 181, 183–187, 189
Therapy .................................................5, 9, 42, 95, 177, 191
Tissue.............................................4, 5, 7, 9, 12, 13, 17, 29, 36, 45, X
46, 55, 56, 58, 62, 68, 80–84, 87, 90, 94, 95, 97, 100–103,
105, 106, 121–123, 167, 177, 178, 180, 181, 183–186, Xeno ...................................................................................68
189, 194, 199
Z
Tomaskovic-Crook, E............................... 151–164, 199–206
Transfection ..............................................................166–170 Zabaleta, L. ..............................................................177–189
Transfer .............................................121, 122, 124–128, 169 zona pellucida ................................................... 123, 124, 128

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What is the book about?

What is the book about?

What are some of the techniques discussed for stem cell banking?

What are some of the techniques discussed for stem cell banking?

Methods in

Molecular Biology 1590

For further volumes:

Concepts and Protocols

ISSN 1064-3745 ISSN 1940-6029 (electronic)

Library of Congress Control Number: 2017934046

© Springer Science+Business Media LLC 2017

Printed on acid-free paper

This Humana Press imprint is published by Springer Nature

Wollongong, NSW, Australia Jeremy M. Crook

PART I GENERIC THEMES IN STEM CELL BANKING

PART II PROTOCOLS FOR PLURIPOTENT STEM CELL BANKING

PART III PROTOCOLS FOR MESENCHYMAL STEM CELL BANKING

PART IV PROTOCOLS FOR HUMAN NEURAL STEM CELL BANKING

IZASKUN BELOQUI • StemTek Therapeutics, Derio, Spain

JOY RATHJEN • School of Medicine, University of Tasmania, Hobart, TAS, Australia

Generic Themes in Stem Cell Banking

Stem Cell Banking: A Global View

1 The Rationale for Stem Cell Banks

In recent work to establish the European bank of iPSC lines (www.

2 History of Banking Human Pluripotent Stem Cell Lines

The discovery that hESCs could be generated from human blasto-

ranging restorative therapy to replace damaged or diseased tissue

Distributor Location Website

3 Core Requirements for the Establishment of a Pluripotent Stem Cell Bank

There are at least three fundamental issues to address before set-

4 Technical Challenges for Stem Cell Banks

In order to deliver the large numbers of existing and future cell

concern is to be expected wherever work on gametes is proposed

Quality Assurance in Stem Cell Banking: Emphasis

Fig. 1 Schematic of quality management in a stem cell bank

total quality management including risk assessments of all aspects of

2 Quality Systems and Standards

Good Laboratory Practice (GLP) regulatory framework is applied

GMP include the quality of management and distribution of the

Fig. 2 A ﬂow chart of QA in a hESC bank

3 Consents and Legal Framework

Despite several sets of guidelines, the main principles for QA of

4 QA Is Necessary for Stem Cell Banking Activities

In order to ensure the banking and provision of good quality cells,

Acquisition and Reception of Primary Tissues, Cells,

There is a wide variety of biological material used in the processing

and iPSCs [7–17]). Such repositories operate within a governance

2 Sourching Material for Procurement and Acquisition

consented for the intended purpose [36–38]. The consent needs

from a recognized supplier. The principles outlined in the consen-

3 Points to Consider with Respect to Procurement from Suppliers of Primary

When sourcing a supplier of biological material it is good practice

A material transfer agreement (MTA) might be required to be

4 Accessioning and Receipt of Biological Materials

As biorepositories, stem cell banks are dedicated facilities, often

On receipt of material into the repository, the material should

The author would like to thank the Medical Research Council

Population-based biobanks aim to recruit high-quality samples from

and/or cells is a prerequisite, the information generated from han-