ATLA 30, 407414, 2002 407

Good Cell Culture Practice

ECVAM Good Cell Culture Practice Task Force Report 1

Thomas Hartung,1 Michael Balls,2 Claudia Bardouille,3,4 Olivier Blanck,5 Sandra Coecke,2
Gerhard Gstraunthaler6 and David Lewis3

1Biochemical Pharmacology, University of Konstanz, 78457 Konstanz, Germany; 2ECVAM, Institute for
Health & Consumer Protection, European Commission Joint Research Centre, 21020 Ispra (VA), Italy;
3European Collection of Animal Cell Cultures, Centre for Applied Microbiology & Research, Porton Down,
Salisbury SP4 0JG, UK; 5Pfizer Central Research, Amboise, Poce-sur Cisse, 37401 Amboise, France;
6Institute of Physiology, University of Innsbruck, Innsbruck, Austria

Background condition for the publication of work involving

cell and tissue culture. This statement should
The maintenance of high standards is fundamental to be reviewed and updated at further World
all good scientific practice, and is essential for ensur- Congresses on Alternatives and Animal Use in
ing the reproducibility, reliability, credibility, accept- the Life Sciences.
ance and proper application of any results produced.
The aim of this Good Cell Culture Practice (GCCP) These initiatives resulted from recognition of the
initiative is to reduce uncertainty in the development rapidly expanding use of in vitro systems, not only
and application of in vitro procedures, by encouraging in basic research, but also to meet regulatory
the establishment of principles for the greater inter- requirements for chemicals and products of various
national harmonisation, rationalisation and stan- kinds. Indeed, some of the recent major develop-
dardisation of laboratory practices, nomenclature, ments in the use of in vitro systems have been in
quality control systems, safety procedures and the fields of toxicity testing and product quality
reporting, linked, where appropriate, to the applica- control, and experience gained here and in the vali-
tion of the principles of Good Laboratory Practice dation of alternative (i.e. non-animal) procedures
(GLP), as recently interpreted for in vitro studies (1). has much to offer to other users of in vitro technol-
The ECVAM Task Force on GCCP was estab- ogy.
lished in response to proposals made at a workshop In vitro systems are also finding increasing appli-
on the standardisation of cell culture procedures cation as production systems for various kinds of
(2), held during the Third World Congress on materials, including monoclonal antibodies, vac-
Alternatives and Animal Use in the Life Sciences cines, hormones, drugs and nutrients, for use in
(Bologna, Italy, 29 August2 September 1999; 3). In research, diagnosis and therapy. In addition, there
a subsequent plenary session, the Congress partici- is an increasing use of genetically modified human
pants as a whole endorsed the following statement: and animal cells, and of cells and tissues derived
from genetically modified animals. New therapies,
The participants in the Third World Congress based on cell and gene therapy and tissue engineer-
on Alternatives and Animal Use in the Life ing, in which in vitro methodologies play a vital
Sciences call on the scientific community to role, are also becoming more widely used.
develop guidelines defining minimum stan- Further significant developments are certain to
dards in cell and tissue culture, to be called result from the following: the use of in vitro systems
Good Cell Culture Practice (GCCP), analogous for high throughput screening; the human genome
to the OECD Principles of Good Laboratory project; the emerging fields of genomics and pro-
Practice (GLP), which cannot normally be fully teomics; and the use of biomarkers of disease, sus-
implemented in basic research, on the grounds ceptibility, exposure and effect.
of cost and lack of flexibility. Such guidelines The in vitro systems themselves are also becom-
should facilitate the interlaboratory compara- ing more varied and more sophisticated, with the
bility of in vitro results. One of the intentions development of co-culture, sandwich culture, perfu-
of this statement is to encourage journals in sion culture, aggregate culture, hanging-drop meth-
the life sciences to adopt these guidelines as a ods, airliquid interface maintenance, long-term

4Present address: Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany.

408 T. Hartung et al.

maintenance, controlled cell differentiation, and Starting Materials

tissue-reconstruction approaches, including organ-
otypic cultures. The cells or tissues used in test systems may be
The ECVAM Task Force on GCCP was estab- freshly isolated from animal or human donors (pri-
lished in the autumn of 1999 under the chair- mary cells) or, at the other extreme, may comprise
manship of Thomas Hartung (University of a laboratory-adapted strain or line that has been
Konstanz, Germany). The remit given to the task serially propagated and maintained in continuous
force was to define the scope of GCCP guidance culture for long periods (continuous cell lines).
and to further its elaboration. The task force has Freshly isolated primary cells will rapidly dediffer-
met three times, in Zurich, Switzerland; entiate in culture, and they have a limited capacity
Innsbruck, Austria; and Ispra, Italy. The propos- to multiply. The ability to proliferate in vitro, as
als and comments presented in this report pro- shown by continuous cell lines, is associated with
vide a basis for the drafting of a GCCP guideline genetic and phenotypic modifications of the type
document at an ECVAM workshop to be held in commonly associated with tumour cells.
the near future. Continuous cell lines are poorly differentiated,
The scope of this report has deliberately been and lose many of the phenotypic characteristics of
broadly defined, to include systems based on cells, the original cell type in vivo. They have a variable
tissues and organs obtained from humans and ani- capacity for serial propagation, and some cell lines
mals, and issues related to: nomenclature; labelling show senescence, and progressively lose the ability
and reporting; characterisation and maintenance of to multiply, typically after 1520 population dou-
essential characteristics; quality assurance; culture blings. The potential for cell-associated variability
media; storage; cell culture collections; human tis- for primary cells is different from that of continu-
sue banks; patenting; education and training; ous cell lines and cell strains (which have a limited
safety; and ethics. life-span of up to 50 population doublings).
The intention of the ultimate guidelines will be to
foster a consensus among all concerned, in any way,
with the use of in vitro systems, in order to assist Primary cell and tissue cultures
scientists involved in research, testing, biotechnol-
ogy, bioreactor production and clinical applications, Primary cultures isolated from animals or humans
to establish and maintain best laboratory practices, represent heterogeneous populations with respect,
to promote effective quality control systems, to for example, to differences in cell types and states of
facilitate education and training, to support journal differentiation. Each isolate will be unique and
editors and editorial boards, to assist research fund- impossible to reproduce exactly. The process of ded-
ing bodies, and to help any authorities who need to ifferentiation commences the moment that cells are
interpret and apply conclusions based on in vitro separated from their parent tissues, so a primary
data. cell culture is a dynamic system in a constant state
of change. Primary cell cultures commonly require
complex nutrient media, supplemented with animal
The Inherent Variation of In Vitro serum and other non-defined components. Con-
Systems sequently, primary cell culture systems are extremely
difficult to standardise.
Cultured human and animal cells are increasingly
used as the basis for simplified, direct test sys-
tems that have the potential to be more control- Continuous cells and cell strains
lable and more reproducible than test systems
employing laboratory animals. However, if a bio- Immortalised cell lines and cell strains are able to
logical test system is simplified to fundamental multiply for extended periods in vitro, and can be
levels, it is paramount that the essential compo- expanded and cryopreserved as cell bank deposits.
nents of such a reduced system are closely defined Most of the fundamental phenotypic changes that
and are reproducible. The standardisation of in occur shortly after the original isolation from the
vitro systems begins with control of the starting parent animal tissue have been completed, so a con-
materials. tinuous cell line is more homogeneous, more stable,
The initiation of an in vitro system essentially and so more reproducible than a heterogeneous
involves the donor, the cells or tissue, the culture population of primary cells. The outstanding disad-
medium, and the substratum. These components vantage of most continuous cell lines is that they
interact, and the properties of the total system and retain little phenotypic differentiation, and poorly
any variation in them are undoubtedly a result of represent the in vivo situation.
this interaction. However, the potential for varia- The opportunity for variability of continuous cell
tion can also be considered for each separate com- lines stems from the very fact that they can often be
ponent. cultured indefinitely. Continuous cell lines have
Good cell culture practice 409

been distributed worldwide in a totally uncontrolled will be communicated to the scientific community
manner, creating a massive potential for identifica- in the form of a scientific publication.
tion errors and the introduction of contaminants. Where the work needs to be carried out routinely,
Therefore, it is recommended that authenticated a procedure should be written down. In the context of
stocks of a continuous cell line are purchased from regulatory acceptance, this procedure should be capa-
a recognised, national or international animal cell- ble of being developed into a proper GLP-compliant,
culture repository, such as the Deutsche Sammlung stepwise Standard Operating Procedure (SOP).
von Mikroorganismen und Zellkulturen GmbH Many guidelines are available concerning what a
(DSMZ) in Germany, the American Type Culture scientific report for academic, industrial or regula-
Collection (ATCC), the Riken Gene Bank in Japan, tory purposes should contain. To achieve a wide
or the European Collection of Animal Cell Cultures acceptance of the GCCP guidelines, it is therefore
(ECACC) in the UK. crucial to encourage implementation of these prac-
Nevertheless, it is possible that even samples of an tices at all levels where cell and tissue culture work
authenticated continuous cell line with the same is conducted and reported.
nominal identity, but purchased from different The recommended content of a GCCP-compliant
sources, may exhibit phenotypic differences that study report, including a study plan and a GLP-
reflect divergence arising from their different culture compliant SOP, should form an integral part of
histories. It is even possible that different banks of GCCP guidelines.
the same cell line, manufactured within the same
repository, may exhibit minor differences that could
be significant in the context of a specific study. Cells

Any report on cell culture experiments should

Culture media include a basic description of the cells cultured,
including, where appropriate, the following.
Cell culture medium usually comprises a defined
base solution, which includes salts, sugars and 1. Nomenclature of cell type or line in use (code,
amino acids, mixed with a variety of supplements, such as ATCC number, alias); the lot number of
depending on the culture requirements of the cell the cell bank represents the ultimate definition
type. Medium supplements, particularly sera from of a cell.
various animal species or from different suppliers,
are inevitably complex and cannot be defined. In 2. a) Origin and mode of culture initiation (species,
fact, all the components of culture media comprise organ, tissue, lineage, mode of transformation,
a significant potential source of variability. genetic modification, sublines/hybrid cells;
A batch of animal serum is often a pool of dona- and, in the case of human cells: donor charac-
tions taken from a large number of animals. Such a teristics, such as race, sex, age, health status,
pooling strategy can result in an acceptable degree medication, disease, biopsy, and tumour sta-
of homogeneity between different batches of animal tus).
serum produced by the same manufacturer.
However, there are likely to be qualitative differ- b) Methods of cell isolation (for example,
ences between sera collected in different geograph- mechanical, enzymic); transport and storage
ical regions. of biopsies and explants.
The concentration/activity of growth factors
must not be expressed in g/ml but in Units/ml 3. Source, such as cell bank (ATCC, ECACC,
where Unit corresponds to a consistent (WHO) DMSZ, Riken Gene bank, etc.) or depositor, and
standard. laboratory of origin, as well as information on
original publication/patent; shipping of cells, i.e.
frozen or in liquid medium.

Reporting on Cells, Other Materials and 4. Basic morphological description of cultured

Equipment cells, including the phenotype and its stability
and expected doubling time.
To produce a high-quality scientific report, various
components have to be incorporated, namely, gen- 5. Culture conditions, including basic medium and
eration of ideas, planning and experimental design supplements/additives, additional buffer sys-
(including statistical aspects), execution of the tems, composition of incubator atmosphere,
study, data collection and analysis, and discussion maintenance temperature.
and conclusions.
The final report is the ultimate goal of all scien- 6. Subcultivation intervals (cell density, conflu-
tific work, which (if not restricted for internal use) ent/subconfluent cultures, cell harvest, split
410 T. Hartung et al.

ratio, initial passage number, number of pas- approximate in vivo-like cell behaviour. In general,
sages in culture). cell proliferation and differentiation are conflicting
aims of cell culture. This implies the following.
7. Conditions for freezing/thawing, including cryo-
protectant, storage conditions, viability, plating 1. The impact of variation of these materials
efficiency. should be monitored and documented.

8. Indicators of the state of differentiation and 2. Equipment and instruments should be properly
expression of specific activities, where available; maintained and calibrated (for example, control
precise definition of measures undertaken to of temperature and CO2 levels of incubators).
maintain or induce differentiation and activity
3. All materials employed should be stored under
9. Test method, dose and test intervals for appropriate conditions to protect them from
Mycoplasma contamination, and other appro- damage, infestation or contamination.
priate controls.
4. Changes of batches of material should be moni-
tored with regard to their influence on principal
Other materials, equipment and procedures endpoints in use in a study.

The quality of equipment and cell culture materials 5. Measures should be taken to assess the status of
should be sufficient to maximise the generation of cells before any experiment is undertaken.
reproducible and reliable results.
6. Suitable measures should be employed for cell
1. The compositions of culture media for routine line identification and verification, and for test-
cultures (maintenance media) and/or experi- ing for cross-contamination.
mental cultures, and supplements/additives (for
example, serum, heat-inactivation or irradia- 7. The differentiation state/phenotype of cells
tion of serum, growth factors, hormones, antibi- should be monitored (for example, by morphol-
otics) should be defined. ogy, histochemistry, enzyme/gene expression,
growth rate, viability, sensitivity to toxins, abil-
2. Non-defined preparations (for example, serum ity to stimulate cell functions, surface markers,
replacements, growth factor mixtures) should adherence to substratum, DNA/chromosome
not be considered acceptable, except in specific analysis, or fingerprinting).
circumstances.
8. The appropriate measure of differentiation
3. Volumes of media used, and feeding cycles should be independent of the cellular function
should be defined. under study, and should be assessed at least at
the beginning and at the end of each series of
4. Culture vessels (for example, flasks, Petri experiments.
dishes, bottles, roller cultures) should be
defined. 9. Positive and negative controls should be
included in all experiments.
5. Culture substrata, coating materials (for exam-
ple, collagen, fibronectin, laminin), and coating The value of studies on cell cultures is endangered
procedures should be defined. by contamination and/or infection. Therefore, lab-
oratory animals from an outside source, to be used
6. Names and addresses of the manufacturer/sup- as donors, should be kept in quarantine for an
pliers of culture media supplements, vessels and appropriate period. Similarly, appropriate meas-
substrata should be given. ures should be taken when a cell line is introduced
into the laboratory, to ensure that no infection/
contamination of cell lines already present can
Quality Assurance occur (certificate from supplier, testing for the
more common contaminants [for example, Myco-
The aim of a quality assurance regimen is to assure plasma], growth on antibiotic-free medium for a
consistency, traceability and reproducibility. In specified period). Tests for frequent contamina-
each laboratory, a person responsible for the qual- tions (such as Mycoplasma) should be performed
ity assurance of cell culture work should be on a regular basis, and results should be discarded
appointed. in the event of any evidence of contamination of
When in vitro systems are used to model the in materials. If an infection has been eradicated, the
vivo situation, all possible efforts should be made to regimen should be defined and reported.
Good cell culture practice 411

The onus is on a laboratory to confirm that Whenever possible, testing for viral infections
incoming materials are suitable for the intended should be performed at the very beginning of
purpose, by: cell/tissue isolation. A primary consideration in
the research use of human tissues is the safety of
1. identifying the critical materials; all the personnel who will be, or could be, exposed
to such tissues, or any product obtained from
2. defining the test and criteria/markers related to them, such as cell cultures or cell fractions. The
suitability; most important aspects of ensuring safety are
awareness, suitable handling and disposal proce-
3. either introducing a regimen to test incoming dures, and the provision of adequate training for
materials/lots for these markers, or obtaining all users. Although it is possible to test for a num-
certification from manufacturers/suppliers; ber of viral and bacterial pathogens, there are sig-
and nificant residual risks when this testing has been
performed. Therefore, such testing does not
4. keeping reference samples of previously absolve either the provider of non-transplantable
used lots for comparison with incoming mate- tissue or the end-user from taking appropriate
rials. precautions against known and unknown hazards.
As the cases of HHV8 and Kaposis sarcoma, and
In addition, it is vital to introduce an adequate sys- of Coxsaeckie 4 and juvenile diabetes have recently
tem of record keeping, and to maintain appropri- demonstrated, new human viruses linked with
ate records concerning all those conducting the pathogenic conditions have been discovered as
work. often as every 510 years, and more are likely to be
found in the future. Therefore, whatever the
results of specific testing, all human tissue must
Safety be considered to be hazardous at all stages of its
procurement, experimental use and disposal.
Some safety issues connected with cell and tissue Personnel should be vaccinated whenever possible
culture are summarised in Table 1. (for example, against hepatitis B). Although the
Potentially infectious materials are widely used, risk for the experimenter may be relatively low
and all work with human materials should be per- during standard procedures, the proper handling
formed in Class II cabinets. and disposal of waste material is critical.

Table 1: Some safety issues connected with cell and tissue culture

Primary cultures Primary cultures of human origin may be a source of viral infection (for example, HIV,
hepatitis B, hepatitis C), and precautions should be agreed with the tissue-supplying agency.
Primary cultures of animal origin may also be a source of infection or allergy.

Cell lines Cell lines may contain endogenous viruses, genetically manipulated material, be of tumour
origin and/or be tumorigenic.

Procedures Procedures should be performed in suitable facilities, according to local legal regulations (for
example, sterile/aseptic working place, use of laminar-flow cabinets and cell culture
incubators).
Experimental procedures and downstream processing of culture should be clearly defined
(for example, cell harvesting, isolation of cell culture products, virus propagation, vaccine
production, induction of differentiation).
Proper handling of liquid nitrogen during cryopreservation of cells and retrieval of vials
from frozen storage is essential.

Disposal All waste should be treated properly, to minimise any threat to humans (for example,
toxicity, mutagenicity, teratogenicity), as well as to other cells and animals under study.

Infections The main safety concern is the potential for worker infection (viruses, bacteria, fungi,
mycoplasmas and parasites are potential pathogens).
Potential exists for continuous cell lines to carry latent viruses and for transformed lines
to spontaneously produce viruses with oncogenic potential in humans.
412 T. Hartung et al.

The assessment of risk requires a knowledge of The number of people directly exposed to any
the history and status of the cell population: risk is risks should be kept to an absolute minimum, and
low when the work involves cells derived from such exposure should be strictly limited to those
pathogen-free animals, or cell lines that have been who have sufficient knowledge and training to be
determined to be free of adventitious agents. The considered informed as to their personal safety.
potential for infection increases when the work All such personnel should be provided with what-
involves the use or the production of pathogenic ever effective and safe immunisations are available
agents. In cells from pathogen-free sub-primate at the time. Tracking methods that clearly identify
species, the principal safety concern is probably the samples of human origin, especially when this is not
potential for injury associated with handling liquid obvious by inspection, for example, tissue
nitrogen during the cryopreservation of cells and homogenates or DNA extracts, which may contain
the retrieval of vials from frozen storage. In labora- viral DNA, are essential, to ensure that the appro-
tories where glass pipettes are used, breakage of priate containment is maintained at all times, up to
these can also pose a significant threat. and including the final disposal and decontamina-
The main safety concern is the potential for infec- tion of all samples.
tion of workers from continuous cell lines carrying Specific procedures that may amplify a hazard
latent viruses, and for transformed lines sponta- and thus greatly increase risk, such as the in vitro
neously to produce viruses with oncogenic potential culture of a susceptible cell type from a potentially
in man. Because human cells and body fluids can infected donor, must be clearly identified, and the
carry infectious agents such as HIV and hepatitis risks posed should be separately assessed before the
viruses, cells of human origin must be considered a work is undertaken.
potential risk. It is important to remember that
HIV has been isolated from human cells and tis-
sues, cell extracts, whole blood, and body fluids, Education and Training
including semen, vaginal secretions, cerebrospinal
fluid, tears, breast milk, and urine. In successful cell and tissue culture, living material
All work with human cells should be carried out from various tissues should proliferate and/or func-
on the assumption that the specimen may carry an tion under appropriate culture conditions, while
infectious agent. Significant viral pathogens that preserving sufficient differentiated characteristics,
could be encountered include hepatitis B, hepatitis which closely resemble those of their ancestors in
C, HIV and cytomegalovirus (CMV), all of which vivo.
have specific known pathogenicities, and the Additional aims can include permitting cells to be
recently identified HHV8 (Kaposis sarcoma virus), manipulated either genetically or chemically, trans-
which may cause tumours under certain circum- fecting cells for the expression of foreign genes and
stances. In addition, the possibility of bacterial haz- proteins, or the use of cultured cells for virus prop-
ards, such as various Mycobacterium species, and agation or vaccine production.
antibiotic-resistant organisms, such as methicillin- Therefore, the proper training of all personnel in
resistant Staphylococcus aureus, should not be basic cell culture practice, in the application of spe-
ignored, as they are becoming increasingly frequent cific procedures, and in general and specific safety
among hospital cases. precautions, depending on the types of cells (pri-
There is also a potential risk that certain human mary cultures, transformed and/or transfected
or primate cell lines, if able to enter the body, may cells, etc.) and on the aims of the work, should be
have oncogenic potential. A classification of cell viewed as mandatory. Some aspects of training are
lines as aetiological agents can be consulted. summarised in Table 2.
The safe use of human tissue that might contain
known and unknown infectious agents depends on
secure containment during collection, transport Ethical Issues
and use (to minimise exposure of laboratory per-
sonnel and transportation couriers who handle From an ethical and a legal point of view, it is desir-
such tissues directly), effective methods of sample able to establish high standards for cell and tissue
decontamination prior to release for disposal from culture worldwide, so that ethical acceptability,
the laboratory (to eliminate risk to the environment safety and accountability can be guaranteed, inso-
and to the general public), and strict protocols for far as that is possible.
handling and disposal as waste. It is essential that Two ECVAM workshops have considered ethical
the experiments are performed in suitable facilities, and quality control aspects related to cells and tis-
according to the legal regulations (sterile/aseptic sues obtained from human donors (4, 5). These
working place, use of laminar-flow cabinets and cell aspects extend from the initial contact with the
culture incubators). Full descriptions of experimen- donor or their next-of-kin, through isolation and
tal procedures and downstream processing of cul- supply procedures, to use in the research facility. It
tures are essential. was strongly felt that human tissues should be sup-
Good cell culture practice 413

Table 2: Aspects of training in the use of 8. Awareness of the widespread use of potentially
basic culture techniques and infectious materials in cell and tissue culture
examples of special culture should be improved.
procedures
9. Many aspects of the patenting of cell lines need
clarification. Access to patented cell lines
Basic culture technique should be regulated and harmonised.
Principles of sterile technique
Handling of culture media 10. The Declaration of Bologna should be updated
Feeding of cultures (media changing)
at future World Congresses on Alternatives and
Cell counting
Subcultivation (trypsinisation) Animal Use in the Life Sciences.
Detection and elimination of contamination
Growth parameters, growth curves
Viability assays Acknowledgement
Storage and freezing/thawing of cells
The authors are grateful for valuable comments
Special culture procedures from Dr L. Steeb/Dr Brcsk (PromoCell,
Primary cell and tissue cultures Heidelberg, Germany), Dr K. Macfelda (AKH Wien,
Toxicity testing, viability assays Austria) and Dr J. Noraberg (Neuroscreen, Odense,
Cloning
Transfection, expression cloning Denmark).
Cell transformation and immortalisation
Virus propagation and isolation
References
1. Cooper-Hannan, R., Harbell, J.W., Coecke, S., Balls,
plied through officially recognised human tissue M., Bowe, G., Cervinka, M., Clothier, R., Hermann,
F., Klahm, L.K., de Lange, J., Liebsch, M. &
banks, and collected according to acceptable ethical
Vanparys, P. (1999). The principles of Good
standards and in compliance with national laws. Laboratory Practice: application to in vitro toxicol-
The members of the ECVAM Task Force on GCCP ogy studies. The report and recommendations of
endorse the recommendations made in the reports ECVAM workshop report 37. ATLA 27, 539577.
on ECVAM workshops 32 (1), 37 (4) and 44 (5). 2. Gstraunthaler, G. & Hartung, T. (1999). Bologna
Declaration toward Good Cell Culture Practice.
Third World Congress on Alternatives and Animal
Use in the Life Sciences, Bologna, Italy. ATLA 27,
Conclusions and Recommendations 206.
3. Hartung, T., Gstraunthaler, G. & Balls, M. (2000).
1. An internationally agreed set of guidelines or Bologna Statement on Good Cell Culture Practice
principles on GCCP should be produced and (GCCP). ALTEX 17, 3839.
published. 4. Anderson, R., OHare, M., Balls, M., Brady, M.,
Brahams, D., Burt, A., Chesne, C., Combes, R.,
Dennison, A., Garthoff, B., Hawksworth, G., Kalter,
2. The guidelines should be adopted by editorial E., Lechat, A., Mayer, D., Rogiers, V., Sladowski, D.,
boards of journals, in order to improve the Southee, J., Trafford, J., van der Valk, J. & van
reporting of essential information. Zeller, A.M. (1998). The availability of human tissue
for biomedical research. The report and recommen-
3. The guidelines should be adopted by research dations of ECVAM workshop report 32. ATLA 26,
763777.
funding bodies, in order to define quality con-
5. Anderson, R., Balls, M., Burke, M.D., Cummins, M.,
trols and ensure the overall quality of in vitro Fehily, D., Gray, N., de Groot, M.G., Helin, H., Hunt,
studies. C., Jones, D., Price, D., Richert, L., Ravid, R., Shute,
D., Sladowski, D., Stone, H., Thasler, W., Trafford,
4. The guidelines should be used in education and J., van der Valk, J., Weiss, T., Womack, C. &
training in the use of in vitro techniques. Ylikomi, T. (2001). The establishment of human
research tissue banking in the UK and several west-
ern European countries. The report and recommen-
5. A harmonised nomenclature system for cell dations of ECVAM workshop report 44. ATLA 29,
lines should be introduced. 125134.

6. The authentication of cell lines according to

standards laid down by independent bodies Further Reading
would be useful.
Arlett, C.F. (2001). Increased use of human cell lines: yet
more skeletons in the cupboard? ATLA 29, 513515.
7. A central registration of cell characteristics Arlett, C.F. (2001). The use of dubious cell lines in
would be useful. research: is trust enough? Lancet Oncology 2, 467.
414 T. Hartung et al.

Bardouille, C. (2001). Maintenance of Cell Cultures (ed. H. Schffl, H. Spielmann, F.P. Gruber, H. Appl,
with special emphasis on eukaryotic cells. In F. Harrer, W. Pfaller & H.A. Tritthart), pp. 275281.
Biotechnology (ed. H-J. Rehm & G. Reed), pp. 2840. Vienna, Austria: Springer.
New York, NY, USA: John Wiley & Sons. Hayflick, L. (1990). In the interest of clearer communica-
Cartwright, T. & Shah, G.P. (1994). Culture media. In tion. In Vitro Cellular and Developmental Biology
Basic Cell Culture: A Practical Approach (ed. J.M. 26, 16.
Davis), pp. 5791. Oxford, UK: Oxford University MacLeod, R.A.F. & Drexler, H.G. (2001). Cell banks
Press. detect false cell lines: journals must act too. Lancet
Darling, D.C. & Morgan, S.J. (1994). Animal Cells: Oncology 2, 467468.
Culture and Media, Essential Data, 151pp. New McAteer, J.A. & Davis, J. (1994). Basic cell culture tech-
York, NY, USA: John Wiley & Sons. nique and the maintenance of cell lines. In Basic Cell
Doyle, A. & Griffiths, J.B. (1997). Mammalian Cell Culture: A Practical Approach (ed. J.M. Davis), pp.
Culture: Essential Techniques, 174pp. New York, 93148, Oxford, UK: Oxford University Press.
NY, USA: John Wiley & Sons. Markovic, O. & Markovic, N. (1998). Cell cross-contami-
Doyle, A., Griffiths, J.B. & Newell, D.G. (1998). Cell and nation in cell cultures: the silent and neglected dan-
Tissue Culture: Laboratory Procedures, Vol. 3. New ger. In Vitro Cellular and Developmental Biology 34,
York, NY, USA: John Wiley & Sons. 18.
Fedoroff, S. (1967). Proposed usage of animal tissue cul- Masters, J.R.W., Twentyman, P., Arlett, C., Daley, R.,
ture terms. Experimental Cell Research 46, 642648. Davis, J., Doyle, A., Dyer, S, Freshney, I., Galpine,
Freshney, R.I. (2000). Culture of Animal Cells: A Manual A., Harrison, M., Hurst, H., Kelland, L., Stacey, G.,
of Basic Techniques, 4th edn, 486pp. New York, NY, Stratford, I., & Ward, T.H. (1999). UKCCCR
USA: John Wiley & Sons. Guidelines for the Use of Cell Lines in Cancer
Froud, S.J. & Luker, J. (1994). Good laboratory practice Research, 52pp. London, UK: United Kingdom Co-
in the cell culture laboratory. In Basic Cell Culture: ordinating Committee on Cancer Research.
A Practical Approach (ed. J.M. Davis), pp. 273286. Anon. (1988). Chancen und Risiken durch Suger-
Oxford, UK: Oxford University Press. zellkulturen. Forum Mikrobiologie 11, 359367.
Gstraunthaler, G. (2000). Standardisierung in der Anon. (1991). Gefhrdungspotential durch Retroviren beim
Zellkultur: wo fangen wir an? In Forschung ohne
Umgang mit tierischen Zellkulturen. BIOforum 11,
Tierversuche 2000 (ed. H. Schffl, H. Spielmann,
428436.
F.P. Gruber, H. Appl, F. Harrer, W. Pfaller & H.A.
Nelson-Rees, W.A., Daniels, D.W. & Flandermeyer, R.R.
Tritthart), pp. 4049. Vienna, Austria: Springer.
(1981). Cross-contamination of cells in culture.
Gstraunthaler, G., Seppi, T. & Pfaller, W. (1999). Impact
Science 212, 446452.
of culture conditions, culture media volumes, and
Petricciani, J.C. (1998). An overview of viral and viral-
glucose content on metabolic properties of renal
epithelial cell cultures. Are renal cells in tissue cul- like agents in cell culture systems. Cytotechnology
ture hypoxic? Cellular Physiology and Biochemistry 28, 4952.
9, 150172. Schaeffer, W.I. (1984). Usage of vertebrate, invertebrate
Hartung, T. & Gstraunthaler, G. (2000). The standardis- and plant cell, tissue and organ culture terminology.
ation of cell culture procedures. In Progress in the In Vitro 20, 1924.
Reduction, Refinement and Replacement of Animal Schaeffer, W.I. (1989). In the interest of clear communi-
Experimentation (ed. M. Balls, A-M. van Zeller & cation. In Vitro Cellular and Developmental Biology
M.E. Halder), pp. 16551658. Amsterdam, The 25, 389390.
Netherlands: Elsevier. Schaeffer, W.I. (1990). Terminology associated with cell,
Hartung, T., Gstraunthaler, G., Coecke, S., Lewis, D., tissue and organ culture, molecular biology and
Blanck, O. & Balls, M. (2001). Good Cell Culture molecular genetics. In Vitro Cellular and Develop-
Practice (GCCP): eine Initiative zur Standard- mental Biology 26, 97101.
isierung und Qualittssicherung von in vitro Spier, R.E. (1998). Animal and plant cell technology: a
Arbeiten. Die Etablierung einer ECVAM Task Force critical evaluation of the technology/society inter-
on GCCP. ALTEX 18, 7578. face. Journal of Biotechnology 65, 111125.
Hay, R.J. (1998). The seed stock concept and quality con- Stacey, G., Doyle, A. & Hambleton, P., eds (1998). Safety in
trol for cell lines. Analytical Biochemistry 171, Cell and Tissue Culture (ed. G. Stacey, A. Doyle & P.
225237. Hambleton), 248pp. Dordrecht, Germany: Kluwer.
Hay, R.J., Reid, Y.A., McClintock, P.R., Chen, T.R. & Taub, M. (1990). The use of defined media in cell and tis-
Macy, M.L. (1996). Cell line banks and their role in sue culture. Toxicology in Vitro 4, 213225.
cancer research. Journal of Cellular Biochemistry Wiebel, F.J., Andersson, T.B., Casciano, D.A., Dickins,
24, Suppl., 107130. M., Fischer, V., Glatt, H., Horbach, J., Langenbach,
Hay, R.J. (1988). The seed stock concept and quality con- R.J., Luyten, W., Turchi, G. & Vandewalle, A.
trol for cell lines. Analytical Biochemistry 171, (1997). Genetically engineered cell lines: characteri-
225237. sation and applications in toxicity testing. The
Hay, R.J. (2000). Cell line authentication and the seed report and recommendations of ECVAM workshop
stock concept. In Forschung ohne Tierversuche 2000 26. ATLA 25, 625639.