INTRODUCTION TO ANIMAL

TISSUE CULTURE
 Learning Outcome
• Define the common terminology related to
animal cell culture
• Describe what is tissue culture and its
applications
• Explain the advantages and disadvantages of
tissue culture

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 What is Tissue Culture
• Tissue Culture is a general term used for the
removal of cells, tissues, or organs from an
animal and their next placement into an
artificial environment condusive to growth.
• It is also known as techniques of keeping
tissues alive and growing in an appropriate
culture medium.

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 Stem and Cancer
Make protein: Cells
commercial scale

Primary human
Antibody and animal cell
production: culture
monoclonals Cell culture:
why do it

Gene therapy Embryo culture

 Tissue Culture
• Cultures can be initiated from
– tissue or organ fragments
– single cell suspensions
 Tissue Culture
• Tissue culture: a generic term for the in-vitro
cultivation of organs, tissues and cells
• Organ culture: maintaining of whole organ or
fragments of tissue
• Cell culture: culture of individual cells from
animal tissues or established cell line in vitro
• Primary culture: cells derived directly from
organ or tissue of the host organism
• Clone: a homogenous population of cells
derived from a single parental cell & are
genetically identical
 What is cell culture?
• A process by which either prokaryotic or
eukaryotic cells are grown under controlled
conditions.
• In practice it has become to refer to the
culturing of cells derived from multicellular
eukaryotes, especially animal cells.
 What is tissue culture?
• The maintenance of fragments of tissue in
vitro.
• Produces clones that have the same
genotype (unless affected by mutation during
culture).
• A generic term denoting tissue organ culture,
explant culture, and cell culture.
History
 • 19th century: Sydney Ringer developed salt
solutions containing the chlorides of sodium,
? potassium, calcium and magnesium suitable for
maintaining the beating of an isolated animal
heart outside of the body.

• 1885: Wilhelm Roux removed a portion of the

medullary plate of an embryonic chicken and
maintained it in a warm saline solution for
several days, establishing the principle of tissue
culture.

• 1902: Gottlieb Haberlandt proposed that single

plant cells could be cultured– Did not culture
them himself.
 • 1907: Ross Granville Harrison demonstrated the in
vitro growth of living animal tissue (explant of
nerve cord from a frog).

• 1911-1912: Burrows and Carrel grow explants from

adult dogs, cats, rats and guinea pigs and also
malignant tissues.

• 1914: David Thompson begins to experiment with

? organ culture from embryonic chicks.

• 1940s and 1950s: Growing viruses in cell cultures

? allowed preparation of purified viruses for the
manufacture of vaccines.

• 1948: Enders, Weller and Robbins - awarded a

Nobel Prize for their discovery of a method of
growing the virus in monkey kidney cell cultures.
• 1952: George Otto Gey established HeLa
cell line from human cervical carcinoma.
• 1962: the first insect cell line was
established in 1962 by Grace.
• 1970s: Recombinant DNA technology
• 1970- Development of laminar flow
cabinets for cell culture, Kruse et al.
• 1976- Totipotency of embryonic stem cells
described by Illmensee and Mintz.
• 1977- Cross-contamination of many cell
lines with HeLa cells confirmed, Nelson-
Rees & Glandermeyer.
 History
• 1983- Regulation of cell cycle and cycling reported by many.
• 1983- Development of reconstituted cell cultures by Bell and
others.
• 1989- Transformation, malignancy, oncogenes reviewed by
Weinberg.
• 1990- Application of cell culture to production of biotherapeutic
agents.
• 1998- Production of cartilage by tissue engineered cell culture by
Aigner et al.
• 2000- Mapping of the human genome.
• 2007- Use of viral vectors to reprogram adult cells to embryonic
state (induced pluripotent stem cells) by Yu et al.
• 2008 and beyond- Era of induced pluripotent stem cells – promises
and challenges.
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 Major Applications
• A lot of field assorted from animal cell culture
such:

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 Applications of Animal Cell Culture
• Cell culture has become one of the major tools used in cell and
molecular biology.
• Cell culture models can replicate the different properties and
functions of the various organs specific cells in in-vitro conditions
and are the key to successfully translating research findings into
real-world medical applications.
• Animal cells are required for the correct post-translational
processing (including glycosylation) of biopharmaceutical protein
products.
• Production of animal cells for use as in vitro substrates in
pharmacological and toxicological studies.

Some of the important areas where cell culture is currently playing

a major role are briefly described.
(1) Gene Therapy

• Done by modify cells outside of the body by Inserting a normal

gene into somatic cells containing a defective gene to cure the
disease caused by that defect.
• Blood, bone marrow, or another tissue can be taken from a
patient, and specific types of cells can be separated out in the
lab.
• The vector containing the desired gene is introduced into these
cells.
• The cells are left, to multiply in the laboratory, and are then
injected back into the patient, where they continue to multiply
and eventually produce the desired effect.

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• Patient-derived cellular gene therapy products: Cells are
removed from the patient, genetically modified (often using a
viral vector) and then returned to the patient.
• Viral vectors - modified viruses can be used as vectors
(vehicles) to carry therapeutic genes into human cells.
• Major targets for this therapy include cancer, HIV, arthritis,
cardiovascular and CNS diseases and cystic fibrosis.

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 Cell Culture: Applications
(2) Model Systems : Cell cultures provide a good model system
for(i) studying basic cell biology and biochemistry, ii)
the interactions between disease-causing agents and
cells, iii) the effects of drugs on cells, iv) the process and
triggers for aging, and v) nutritional studies.

(3) Toxicity Testing: Cultured cells are widely used alone or in

conjunction with animal tests to study the effects of new
drugs, cosmetics and chemicals on survival and growth in a
wide variety of cell types. Especially important are liver-
and kidney-derived cell cultures.
 Cell Culture: Applications
(4) Cancer Research: Since both normal cells and cancer cells can be
grown in culture, the basic differences between them can be closely
studied. By the use of chemicals, viruses and radiation, to convert
normal cultured cells to cancer causing cells. Thus, the mechanisms
that cause the change can be studied. Cultured cancer cells also
serve as a test system to determine suitable drugs and methods for
selectively destroying types of cancer.

(5) Virology : One of the earliest and major uses of cell culture is the
replication of viruses in cell cultures (in place of animals) for use in
vaccine production.
• Cell cultures are also widely used in the clinical detection and isolation of
viruses, as well as basic research into how they grow and infect organisms.
•For examples: Cell lines such as Vero (Cercopithecus aethiops, Kidney),
293 T(Homo sapiens, Embryonic Kidney), A549 (Homo sapiens,
Lung Adenocarcinoma Epithelial Type II Cells), Caco-2 (Homo sapiens,
Colorectal adenocarcinoma Epithelial Cells) have been used for Drug
Screening, Vaccine/Antibodies Production, Immune Response/Genes
Expression study and Virus Isolation.
 Cell Culture: Applications
(5) Tissue engineering
• To study the sequential or parallel combination of various cell
types to generate artificial tissues (e.g. skin).
• It is a science that combines stem cells, scaffolds with suitable
growth factors, cytokines and chemokines to improve, replace
or regenerate tissues and organs.
• This translational approach has been applied to develop and
design patient-specific tissue grafts that mimic the functional
properties of native tissues.

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• Tissue engineering or organogenesis for production of
bioartificial organ replacement/assisting devices, for example;
• Artificial skin, for severe burns
Tissue engineered bones, to restore skeleton function
Liver assist devices, for hepatic failure
Pancreatic islet of Langerhans devices, for diabetes.
• Cell culture is used for large scale ex-vivo expansion of stem
cells for research and practice of transplantation.
• Stem cells can be categorized broadly into embryonic and
adult stem cells and are efficient cell sources for tissue
regenerative applications.
• The ability of stem cells to expand and differentiate into
desired tissue types makes them an attractive alternative cell
source for regenerative medicine.

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 Cell Culture: Applications
(6) Production of biologicals
– To synthesize valuable biologicals from large
scale cell cultures.
• Monoclonal antibodies
• Hormones
• Enzymes
• Vaccines

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Applications-Production of
Biologics
• Animal cell systems are the preferred "cell factories" for
the production of complex molecules and antibodies for
use as prophylactics, therapeutics or diagnostics.
Examples of these products include;
• o Interferons
o Erythropoetin
o Interleukins and other cytokines
o Tissue type plasminogen activator (tPA)
o Monoclonal antibodies
o Vaccines
Human insulin became the earliest recombinant
protein to be approved as a therapeutic agent,
 Applications
• Monoclonal antibodies- Antibodies that are identical
because they were produced by one type of immune cell.
• Hybridoma technology is a well-established method to
produce monoclonal antibodies (mAbs) specific to antigens
of interest. Hybridoma cell lines are formed via fusion
between a short-lived antibody-producing B cell and an
immortal myeloma cell.
– Applications:
1. Cancer immunotherapy
 Cancer immunotherapy
– Monoclonal antibodies bind only to cancer cell-
specific antigens and induce an immunological
response against the target cancer cell.
– Monoclonal antibodies could be modified for
delivery of a toxin, radioisotope, cytokine or
other active conjugate.
Bispecific Monoclonal antibodies
A bispecific monoclonal
antibody (BsMAb, BsAb) is an artificial protein
that is composed of fragments of two
different mabs and consequently binds to two
different types of antigen. The most widely
used application of this approach is in cancer
immunotherapy, where BsMAbs are
engineered that simultaneously bind to
a cytotoxic cell (using a receptor like CD3) and
a target like a tumour cell to be destroyed.

Antibody-Dependent Cell-Mediated
Cytotoxicity (ADCC) is a mechanism of cell-
mediated immune defense whereby an effector
cell (NK cells, macrophages, neutrophils )of
the immune system actively lyses a target cell,
whose membrane-surface antigens have been
bound by specific abs.
 Chimeric and humanized antibodies
• Human immune system recognizes mouse antibodies
as foreign and rapidly removes them from circulation
and causing systemic inflammatory effects.
• Solution: Merge DNA that encodes the binding
portion of monoclonal mouse antibodies with
human antibody producing DNA, and express it using
mammalian cell culture.
 Monoclonal antibodies drugs approved
• Transplant rejection  Cancer
– Muronomab-CD3  Rituximab
– Daclizumab  Trastuzumab
– Basiliximab
 Gemtuzumab ozogamicin
• Cardiovascular disease
 Alemtuzumab
– Abciximab
 Ibritumomab tiuxetan
• Inflammatory diseases
 Cetuximab
– Infliximab
 Bevacizumab
– Eculizumab
 Tositumomab
– Omalizumab
 Viral infection
– Efalizumab
– Adalimumab  Palivizumab
Applications-Production of Biologics
• Hormone: a chemical messenger from one cell to
another.
• Medication:
1. Thyroxine (hypothyroidism)
2. Steroids (autoimmune diseases, respiratory
disorders)
 Animal cells and Pharmaceutical industry

• Vaccine: an antigenic preparation used to produce active

immunity to a disease, in order to prevent the effects of
infection by any natural or “wild” strain of the organism.
• Vaccines may be living, weakened strains of viruses or
bacteria.
• Vaccines may be killed or inactivated organisms or purified
products derived from them.
• Established animal cells, such as Vero, Madin Darby canine
kidney (MDCK) or chicken embryo fibroblasts (CEFs), are still
the main cell lines used for viral vaccine production.
 Vaccination
• Smallpox
• Rubella
• Polio
• Measles
• Mumps
• Chickenpox
• Flu
 Types of cells
• Anchorage dependent
– Fibroblast
– Epithelial cell
– Muscle cell
– Neuron
• Suspension
– Hematopoietic cells
 Cell morphologies vary depending on cell type

Fibroblastic Epithelial

Endothelial Neuronal
 TYPES OF CELLS
• The adherent cells are typically derived from
tissues of organs such as kidney, liver, heart
where they are immobile in addition to
embedded in connective tissue.
• Each and every one suspension cultures are
derived from cells of the blood system for the
reason that these cells are furthermore
suspended in plasma in vitro e.g. lymphocytes

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Categories of cell cultures based on
origins
 Cell line
• Cell arise from a primary culture after first
successful sub-culture
• 2 categories
– Primary: cells (culture) derived directly from
organ or tissue of the host organism-limited
passage. Normally having the finite number of
passage (< 70 times)
– Continuous: cell having the apparent potential to
be passage indefinitely
 PROCEDURE OF CELL CULTURE
• Primary culture– freshly isolated from tissue
source. Limited passage.
• Cell line – culture that has been passaged
– Finite cell line: dies after several sub-cultures
– Continuous cell line: transformed ‘immortal’. Cell
having the apparent potential to be passage
indefinitely.

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 Cell strain (cell homogenous)
• Cells derived from a primary culture or a cell
line by selection or cloning of cells having
specific properties or markers
• Normally are genetically identical whereby the
culture contains only 1 type of cells
 Cell cloning
• Technique in getting pure cell line/ strain
• Adopt from traditional microbiological approach to
the problem of culture
• Technique used in selecting a hybridoma clone
producing monoclonal antibody
• Several approaches
– Limiting dilution: multiwell technique
– Semisolid agar
– Cloning ring technique: anchorage dependent cell
Passaging or sub-culture

Cell dissociated from flask

Split 1 in 2
Advantages & limitations of animal
cell/tissue culture
 Advantages of cell/tissue culture
• Control of physiochemical environment (pH,
temperature, osmotic pressure, O2 and CO2 tension)
• Can look at direct effect of a molecule on a cell type
• Homogeneity of cell type
• Reduction of animal use
• Large quantities of cells can be obtained
• Economy
• Mechanization (robotics)
 Limitations of cell/tissue culture
 Necessary expertise
 Handling, chemical contamination, microbial contamination, cross
contamination.
 Environmental control
 Workplace, incubation, pH control, containment and disposal of
biohazards.
 Quantity and cost
 Capital equipment, consumables, medium, serum, plastics.
 about 10 times more than using animal tissue.
 Genetic instability
 Heterogeneity, variability.
 Phenotypic instability
 Dedifferentiation, adaptation, selection.
 Identification of cell type
 Expression of markers
 Cell Culture is a Fussy Discipline
• In the tissue culture laboratory:
• bench tops should be kept clear and clean
• wearing a long sleeve lab coat : minimises
contamination from street clothing (hair, etc)
• wearing gloves while doing tissue culture work:
minimises contamination from skin organisms
• Surfaces, gloves, solutions and plasticware sprayed
with 70% alcohol before placed into the biological
hood
• solutions, reagents and glassware used in tissue
culture work should not be shared with non-tissue
culture work
CELL CULTURE POSSIBLE PROBLEM
• Protection of aseptic condition is one of the
most complex challenges in tissue culture
there are quite a lot of route to contamination
which includes malfunction in the sterilization
procedures used for glassware & pipettes,
particulates cross contamination of air inside
the room, weakly maintained incubation,
inappropriate handling.

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 Summary

Cell culture is an invaluable tool for investigators in

numerous fields.
It facilitates analysis of biological properties and
processes that are not readily accessible at the level
of the intact organism.
Successful maintenance of cells in culture, whether
primary or immortalized, requires knowledge and
practice of a few essential techniques.
TERIMA KASIH / THANK YOU
www.upm.edu.my