Guidance On Good Cell Culture Practice

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Good cell culture practices
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ATLA 33, 261–287, 2005 261
Guidance on Good Cell Culture Practice
A Report of the Second ECVAM Task Force on Good Cell Culture

Practice
Sandra Coecke,1 Michael Balls,2 Gerard Bowe,1 John Davis,3 Gerhard Gstraunthaler,4 Thomas
Hartung,1 Robert Hay,5 Otto-Wilhelm Merten,6 Anna Price,1 Leonard Schechtman,7 Glyn
Stacey8 and William Stokes9
1ECVAM, Institute for Health & Consumer Protection, European Commission Joint Research Centre, Ispra
(VA), Italy; 2FRAME, Nottingham, UK; 3Research and Development Department, Bio-Products Laboratory,
Elstree, Herts., UK; 4Department of Physiology, Innsbruck Medical University, 6010 Innsbruck, Austria;
5ATCC, Manassas, VA, USA; 6Généthon, Evry, France; 7National Center for Toxicological Research, Food
and Drug Administration, Rockville, MD, USA; 8Division of Cell Biology and UK Stem Cell Bank, National
Institute for Biological Standards and Control, Blanche Lane, Potters Bar, Herts., UK; 9National Toxicology
Program Interagency Center for the Evaluation of Alternative Toxicological Methods Environmental
Toxicology Program, National Institute of Environmental Health Sciences, Research Triangle Park, USA
Scope of the Report Sciences, Bologna, Italy, in 1999 (2). The proposal that
guidelines should be developed to define minimum
The maintenance of high standards is fundamental to standards in cell and tissue culture, to be called Good
all good scientific practice, and is essential for max- Cell Culture Practice (GCCP), led to the publication of
imising the reproducibility, reliability, credibility, outline guidance on GCCP in 2002 (3). The principles
acceptance and proper application of any results proof GCCP are analogous to the OECD Principles of
duced. The aim of this Guidance on Good Cell Culture Good Laboratory Practice (GLP), which cannot nor-
Practice (GCCP) is to promote the maintenance of mally be fully implemented in basic research, includ-
these standards and to reduce uncertainty in the ing in vitro studies (4).
development and application of animal and human In October 2003, a new task force was convened in
cell and tissue culture procedures and products, by Ispra, Italy, with a broader range of expertise in cell
encouraging greater international harmonisation, and tissue culture, in order to produce a more-detailed
rationalisation and standardisation of laboratory GCCP guidance document which could be of practical
practices, quality control systems, safety procedures, use in the laboratory.
recording and reporting, and compliance with laws, This Guidance is required to serve the rapidly
regulations and ethical principles. expanding use of in vitro systems: in basic research, to
The scope of the document has deliberately been meet regulatory requirements for chemicals and prod-
broadly defined, to include systems based on cells and ucts of various kinds; in the manufacture of various
tissues obtained from humans and animals, and products; in medical diagnostics; and in therapeutic
issues related to the characterisation and mainte- applications such as tissue engineering, and cell and
nance of essential characteristics, as well as quality gene therapy.
assurance, recording and reporting, safety, education Further significant developments are certain to
and training, and ethics. result from, inter alia: the use of in vitro systems for
high throughput screening in pharmacology and toxi-
cology; the human genome project; the emerging
Background fields of genomics, proteomics and metabonomics; and
the use of biomarkers of disease, susceptibility, expo-
The first ECVAM Task Force on GCCP was estab- sure and effect.
lished in the autumn of 1999, in response to proposals This Guidance is intended to support best practice
made at a workshop on the standardisation of cell cul- in all aspects of the use of cells and tissues in vitro,
ture procedures (1), held during the 3rd World and to complement, but not to replace, any existing
Congress on Alternatives and Animal Use in the Life guidance, guidelines or regulations.
Address for correspondence: Sandra Coecke, European Centre for the Validation of Alternative Methods (ECVAM),
Institute for Health and Consumer Protection, European Commission Joint Research Centre, Via Fermi 1, 21020 Ispra
(VA), Italy.
E-mail: sandra.coecke@jrc.it
262 S. Coecke et al.
The Principles of GCCP involving the derivation of new cell lines should
always include detailed records of the derivation
Based on review by a broad range of experts and process and of the reagents and materials used, as
organisations, the aim of this Guidance is to foster the cells may go on to be used for purposes not
consensus among all concerned with the use of cell anticipated at the time, and in some cases, this may
and tissue culture systems, in order to: include clinical use. Particular care should be taken
where cells and tissues are to be used as a reference
— establish and maintain best cell and tissue culture point or reference material, especially for data
practice; interpretation related to the equivalent cells and
— promote effective quality control systems; tissues in vivo.
— facilitate education and training;
— assist journal editors and editorial boards;
— assist research funding bodies; and Critical testing procedures
— facilitate the interpretation and application of
conclusions based on in vitro work. In diagnostics, toxicology and pharmacology, specific
regulations are in place in order to protect human
This GCCP Guidance is based upon the following six health and the environment, such as European
operational principles. Pharmacopoeial requirements and EU and OECD
test guidelines. This Guidance has been written to
1. Establishment and maintenance of a sufficient ensure that the appropriate standards can be main-
understanding of the in vitro system and of the tained when cells or tissues are used in meeting these
relevant factors which could affect it. regulations and requirements.
2. Assurance of the quality of all materials and meth-
ods, and of their use and application, in order to Manufacture of products and therapeutic
maintain the integrity, validity, and reproducibil- preparations of cells and tissues
ity of any work conducted.
A number of specific regulations and requirements
3. Documentation of the information necessary to
relate to the nature and quality of cells and tissues
track the materials and methods used, to permit
used in the manufacture of products, including vac-
the repetition of the work, and to enable the target
cines, monoclonal antibodies, hormones, and cells
audience to understand and evaluate the work.
and tissues for therapeutic use, as well as to the
4. Establishment and maintenance of adequate preparation of the final products. The application of
measures to protect individuals and the environ- GCCP must be consistent with these regulations and
ment from any potential hazards. requirements, and provides guidance generally not
covered in requirements for specific applications.
5. Compliance with relevant laws and regulations,
and with ethical principles.
Principle 1: Establishment and
6. Provision of relevant and adequate education and maintenance of a sufficient
training for all personnel, to promote high quality
work and safety.
understanding of the in vitro system
and of the relevant factors which
could affect it
The Application of GCCP
The essential elements for assuring reliable and accu-
GCCP sets the minimum standards for any work rate work when using cell and tissue-based systems,
involving cell and tissue cultures. However, its are:
detailed implementation depends on the nature of the
work involved. Whilst this guidance is considered a — authenticity, including identity of the system, for
minimum standard for the preparation and mainte- example, provenance and confirmation of geno-
nance of cell cultures, deviations from its specific ele- typic and/or phenotypic characteristics;
ments may be necessary under certain conditions, in — purity, for example, freedom from biological con-
which case they should be justified. tamination; and
— stability and functional integrity of the system in
relation to its intended use.
Research and development
The standardisation of in vitro systems begins with
This guidance is important for research work, to the original animal or human donor and the cells or
avoid poor reproducibility of data and the invalida- tissues derived, and also embraces their subsequent
tion of results from cell culture processes. Research manipulation, maintenance and preservation.
Second ECVAM Task Force on good cell culture practice 263
Standardisation is a difficult task, since cells and Primary cultures and early passage cultures
tissues are prone to change in culture, and
inevitably are subjected to physical and/or chemical The initial in vitro culture of harvested cells and
insults during their isolation, culture, use and stor- tissues taken directly from animals and humans is
age. However, by establishing a framework of pro- called primary culture. In many cases, such cul-
cedures for factors that can be controlled, variation tures also exhibit key characteristics similar to
and other adverse effects on reproducibility and those seen in vivo, so they are widely used for
reliability can be minimised. The availability of basic research and for a number of in vitro appli-
well-characterised and quality-controlled stocks of cations.
cells and tissues, and of media and other critical Although cells in some primary cultures can pro-
reagents, further reduces variability. liferate and can be subcultured (as early passage
Various classifications have been published, cultures), they generally have a limited life-span
which define different types of in vitro cell and tis- and are known to change their differentiated char-
sue systems (see Figure 1 and, for example, refer- acteristics with time in culture. They commonly
ence 5). Three broad categories will be considered require complex nutrient media, supplemented
in this Guidance: with animal serum and other non-defined or ill-
defined components, although serum-free medium
— isolated organs or tissues; formulations are becoming increasingly available.
— primary and early passage cultures; and Primary cultures often represent heterogeneous
— cell lines (including finite, continuous and stem cell populations, and are difficult to standardise and
cell lines). to reproduce, because of uncontrollable variations
between preparations.
Primary cultures have traditionally been main-
1.1 Cells and Tissues tained either in suspension or, more commonly, as
Isolated organs or tissues

Figure 1: Relationships between the main
types of in vitro systems
Isolated organs and tissues, taken for direct use
from animal or human donors, are used for a wide
variety of in vitro applications. These systems are
difficult to standardise, because they often have
Tissue or organ fragment
complex environmental and nutritional needs, and
because of variation between donors.
Tissues or organ fragments can be used, often
perfused with physiological buffers, in a variety of
devices. Such in vitro systems, including isolated Transfer to culture
skin and eye models, are very popular for toxico-
logical applications, due to their similarity with
Dissociated cells
the in vivo situation. It is important to be able to Organ explants
(attachment and
study an adequate number of replicates in such (cell outgrowth)
proliferation)
experiments, and one approach is to use slice tech-
nology. Ultra-thin slices of tissues such as liver,
Primary culture
lung, kidney or brain, can be used to provide a
preparation retaining some of the structural and
functional features of the original organ.
Inevitably, however, such features tend to be rap-
idly lost. Subculture (passage)
Methods involving the isolation and reaggrega-
tion of cells from organs such as the skin, brain and
liver, can lead to the reconstruction of three-dimen-
sional structures, again with some of the structural
and functional properties of the original organ or Cell line
tissue.
Cells from blood and other body fluids are readily
prepared as homogeneous preparations, which are
very useful for in vitro studies. Preparations such
as umbilical cord blood and bone-marrow offer rich
sources of stem cells, and could become the basis of Continuous Finite Stem cell
an expanding range of other systems.
monolayers on glass or plastic surfaces. However, result of sub-optimal in vitro maintenance, han-
methods employing extracellular matrix compo- dling and preservation, is a significant risk for in
nents, and innovative techniques such as the co-cul- vitro cell-based methods.
ture of different cell types and three-dimensional Continuous cell lines may arise spontaneously, or
culture, now offer much greater potential for main- can be produced by using a variety of other method-
taining differentiated structure and function. ologies, such as:
— exposure of normal cells and tissues to irradia-

Cell lines tion and/or treatment with chemical mutagens
or carcinogens;
Cell lines comprise cells that are able to multiply for — isolation from cultures infected with viruses (for
extended periods in vitro and can therefore be example, Epstein-Barr virus);
maintained by serial subculture. They can be subdi- — genetic modification of cells by transfection with
vided into finite cell lines, continuous cell lines and cloned genes (for example, SV40 large T-antigen,
stem cell lines. adenovirus E1, telomerase); and
— isolation from transgenic animals.
Finite cell lines
Finite cell lines are cultures of cells that possess the Stem cell lines
ability to be subcultured numerous times, but Stem cell lines, such as embryonic and germ cell
which eventually cease replication and enter a state lines, are types of continuous cell lines that retain
of senescence, in which cell division has stopped, the characteristics of stem cells and can produce
but the cells remain viable and may also retain diverse differentiated cell types. They require great
some functional activity. care in their maintenance, handling and preserva-
Finite cell lines have a useful life-span in vitro, tion, in order to ensure that their stem cell charac-
and can be maintained as well-characterised and teristics and capacity for differentiation are retained.
quality-controlled cell banks. However, changes Embryonic stem cell lines are usually established
occur as they approach senescence, so they should and maintained on embryonic mouse fibroblasts or
not be used above defined population doubling lim- other feeder cell layers, which are critical to their
its, which can be established by experimental inves- successful culture. Although serum-free and feeder-
tigation. free culture methods are currently being developed,
Numerous finite cell lines have been established. the effects of these new developments on the stabil-
Many of them are human diploid fibroblast cell ity and quality of the cultures have yet to be ascer-
lines, which are genetically stable and remain tained.
diploid for many passages, but which generally Some continuous cell lines, notably cancer cell
reach senescence after 60–70 population doublings. lines, are known to contain stem cell or precursor
cell populations. For the purposes of this
Continuous cell lines Guidance, these are not included as stem cell lines.
Certain cell lines show an apparent ability to be The exact nature and significance of the apparent
subcultured indefinitely, and are known as continu- stem cell component in such lines remains to be
ous cell lines. They do not show the senescence determined.
experienced with finite cell lines. Continuous cell
lines are typically derived from tumours or normal Standardisation for specific uses
embryonic tissues. Standardisation of cell lines used for specialised stud-
While many continuous cell lines have proved to ies and for production purposes will require attention
be stable over long-term passage in vitro, they may to specific characteristics, as well as to the funda-
undergo substantial and irreversible changes. It is mental issues which apply to all cell cultures (see
therefore important to avoid subjecting cell lines to GCCP Principle 2). They should be checked, and
variable culture and passage conditions, and to rechecked at appropriate times, for the expression of
establish cryopreserved stocks of early passage critical functions and markers (for example the path-
cells. ways for biotransformation of xenobiotics, specific
Some continuous cell lines can be a heteroge- cytoskeletal markers, and characteristic morphology
neous mixture of phenotypes (for example, human and ultrastructure). The number of passages for
promyelocytic HL-60 leukaemia cells, RD, SH5Y- which they remain usable should be established.
SY). Other cell lines may undergo changes to the
differentiation state due to certain medium addi-
tives (for example, retinoic acid, dimethylsulphox- 1.2 In Vitro Culture Conditions
ide) or culture conditions (for example, when
adherent cultures, such as Caco-2 or MDCK, are Cell and tissue culture environments differ in many
allowed to reach confluency). In such cases, the respects from those found in vivo. Key elements of
potential for the selection of certain cell types as a in vitro culture conditions include culture media,
supplements and other additives, culture-ware, and Animal sera are a potential source of microbio-
incubation conditions. logical contaminants, notably mycoplasma, bovine
viruses, and possibly the agent which causes Bovine
Spongiform Encephalopathy (BSE). Suppliers use a
Basal medium variety of techniques, including filtration, irradia-
tion and heat-inactivation, to reduce microbial con-
In vitro work is generally performed in complex nutri- tamination. Nevertheless, it is wise, and for some
tive media. Depending on the circumstances, the basal applications, obligatory, to specify sourcing of
culture medium can be serum-supplemented (as in serum from countries where there is a low risk of
traditional cell culture methods) or serum-free, but infection, and, in the case of bovine sera, from ani-
supplemented with additives necessary for obtaining mals of less than 30 months old.
satisfactory cell proliferation and production, or for The use of human serum is restricted to spe-
maintaining a desired differentiation status. cialised applications, as it carries additional risks,
Many slightly different formulations exist under the such as the potential presence of human pathogenic
same general medium names, such as Minimum viruses. Its use must be subject to the strictest qual-
Essential Medium (MEM), and even subtle changes in ity controls, including documentation to demon-
the medium formulation can substantially alter the strate origin and viral safety.
characteristics of certain cells and tissues. In many Because of the disadvantages inherent in the use
cases, these variations are deliberate for specific appli- of animal and human sera, there have been many
cations. Therefore, the medium to be used should be attempts to find alternatives. These have included
precisely specified, and it is important to check that the use of poorly defined supplements (for example,
new supplies of medium meet the required specifica- pituitary extracts, chick embryo extracts, bovine
tions. milk fractions, bovine colostrums), and various plant
extracts (for example, vegetal serum). In some cases,
it is possible to use fully chemically defined media
Serum with appropriate hormones and growth factors. A
compilation of commercially available serum-free
Serum is essential for the maintenance and/or pro- media was published recently, and can be found at
liferation of many cell types. It is a complex mixture http://www.focusonalternatives.org.uk.
of a large number of constituents, including low and
high molecular weight biomolecules with a variety
of physiologically balanced growth promoting and Nutritional status
growth inhibiting activities. However, due to its
complexity and to batch-to-batch variation, serum The exhaustion or inactivation of essential nutri-
introduces unknown variables into a culture system ents in cell culture media, and rising levels of
and can interfere with its performance. metabolites, will inhibit cell growth and cell func-
Animal serum can be derived from adult, new- tion, and will ultimately cause cell death. Planning
born or fetal sources. Bovine sera are most com- an appropriate procedure for medium replenish-
monly used, and during the last few decades, fetal ment (i.e. frequency and volume of medium) and
bovine serum (FBS) has become the standard sup- passaging (for example, split ratio) is therefore
plement for cell culture media. It is a cocktail of essential. This should also be considered when
most of the factors required for cell proliferation using conditioned medium from one culture in an
and maintenance, and thus is an almost universal attempt to promote the growth of another.
growth supplement.
As the composition of serum is highly variable, it
is important that, when an existing batch of serum Antibiotics
is substantially depleted, a new set of serum
batches should be evaluated in parallel with the It is important to remember that antibiotics are
current in-use batch. A range of growth promotion agents that arrest or disrupt fundamental aspects
tests can be used for this purpose, one of the most of cell biology, and, while they are effective against
convenient and most widely used of which is the prokaryotic cells (i.e. bacteria), they are also capa-
plating efficiency test (see reference 6). ble of causing toxic effects in animal cells. Not sur-
It may also be useful for individual users to define prisingly, antifungal agents, being directed at
serum specifications that meet their particular higher order, eukaryotic micro-organisms, are
needs, including the maximum acceptable levels of likely to be more toxic to animal cell cultures. Given
serum components, such as immunoglobulins these obvious contra-indications, the use of antibi-
(which may have inhibitory effects), endotoxins otics in cell and tissue culture should be focused in
(indicative of bacterial contamination, but which two areas: a) protection of tissues, organs, primary
are also powerful cell mitogens), and haemoglobin cultures and cell lines from contamination; and b)
(indicative of haemolysis during clotting). the positive selection of recombinant cell clones
based on the expression of antibiotic resistance inhibit cell growth and may result in cell death. Oxygen
genes. In addition, it is important to obtain antibi- levels may need to be optimised for particular purposes,
otics from companies that are willing to provide cer- for example, to promote growth in large-scale cultures
tification for the concentration and purity of the in bioreactors. For many cell cultures, the appropriate
antibiotics they supply. atmosphere would be 5% v/v carbon dioxide in air, but
Where possible, the use of antibiotics should be the optimum carbon dioxide concentration will depend
avoided. It should not become routine in the cell and on the medium in use, the cells being cultured, and pos-
tissue culture laboratory, and can never be relied on sibly on other specific considerations.
as a substitute for effective aseptic techniques.
pH
Cell culture surface/matrix
The optimal physiological pH for mammalian cell cul-
The surfaces to be used for cell cultures may need to tures is usually considered to be pH 7.2–7.4, and pH
be pre-washed or pre-treated, for example, to achieve 6.0 for insect cells. Variation outside a relatively nar-
the comprehensive wetting of a complex matrix. row pH range may have significant effects on cell phe-
Where coating materials are used, the preparation notype, growth and viability.
method may lead to toxic conditions (for example, low
pH), and washing before cell seeding may be neces-
Cell detachment and subculture
sary. There may be batch-to-batch variation in coat-
ings of biological origin, so pre-use testing is essential.
Detachment solutions, such as trypsin/EDTA, can
have significant effects on cells, if their use in specific
circumstances is not appropriate. Residual detach-
1.3 Handling and Maintenance
ment solutions can lead to adverse effects, and there-
fore should be removed after cell dissociation.
Care should be taken not to expose the cells or tissues Most cell lines are subcultured before they reach
to inappropriate conditions (for example extended confluency. This may be particularly important in
periods out of the incubator). Key items of equipment, some cases, such as where cell differentiation
including incubators, laminar air flow and microbio- occurs progressively after confluency is reached (for
logical safety cabinets, and cryostorage systems, must example, Caco-2 cells). The repeated passage of
be set up and used appropriately (see Appendix 1 and some cell lines after they have reached full conflu-
Appendix 2). ency, may result in the loss of desired characteris-
Aseptic techniques, where appropriate, should be tics. For example, the subculture regime can affect
rigorously applied. The routine isolation, handling and the apparent productivity of recombinant cell lines,
maintenance protocols for cells and tissues should be and the differentiation capacity of Caco-2 cells.
established as Standard Operating Procedures (SOPs).
1.4. Cryopreservation
Temperature
Cells and tissues can be cryopreserved in a stable
The optimal culture temperature depends on the state for limited or prolonged periods. The cryo-
type of cells involved. Insect cells have a relatively preservation process includes freezing, storage and
low optimal growth temperature compared to mam- recovery. In the development of a preservation pro-
malian cells, and their growth characteristics may cedure for a new cell culture, the following points
be altered at higher temperatures, for example, relating to the biochemical and morphological
above 28°C. The exposure of mammalian cells to nature of the culture system, must be considered:
temperatures above 39°C may induce apoptosis,
whilst growth below 35°C may slow replication but — original cell or tissue type (i.e. gross morphology
may also enhance the expression of certain cell pro- or complexity of culture system);
teins. Recombinant cell lines expressing the tem- — growth phase (usually, cells should be harvested
perature-sensitive form of SV40 large T-antigen, during exponential growth to increase the propor-
will replicate at around 33°C, but not at 37°C. tion of cells with a high nucleus:cytoplasm ratio);
and
— status of cells (other biochemical or morphological
Atmosphere features, affected by differentiation, adherence,
etc., will influence the success of cryopreservation).
Oxygen and carbon dioxide are known to be vital for
cell growth, and variations in the levels of these gases There are also a number of key technical elements
can have significant effects on cell cultures. High levels in the process of cryopreservation that should be
of both gases will be toxic, and very low levels will considered, including:
— cryoprotectant (select type and concentration to ple, Epstein-Barr virus from the B95-8 cell line, and
balance the degree of cryoprotection against any human T-lymphotrophic virus II from MT4 cells).
toxic effects, for example, 10% v/v DMSO); Animal viruses are expressed by some cell lines (for
— additives to improve cell survival (for example, example, bovine viral diarrhoea virus in certain bovine
serum); cell lines). Mammalian genomes contain many retro-
— cooling rate (for example, freezing at controlled virus-like sequences, which, whilst not overtly infec-
rate in the presence of the selected cryoprotec- tious, may be released in large quantities as
tant: typically 1°C/minute with 10% v/v DMSO); retrovirus-like particles in murine myeloma cells,
— storage conditions (sufficiently low temperature hybridomas and other cell lines (for example, CHO
to eliminate biological changes, for example, liq- cells and BHK cells). The expression of such virus-like
uid nitrogen vapour or liquid phase); and sequences is also observed at the RNA level in many
— recovery method (for example, rate of thawing, human cancer cell lines and also in primate cell lines.
gradual dilution to minimise osmotic shock, Cross-contamination of cell lines with other cell
removal of cryoprotectant to avoid any toxic lines is a real, but often neglected, problem.
effects). Whenever possible, cells should be obtained from
certified sources, and appropriate procedures
Storage in the liquid phase of nitrogen provides the should be applied to minimise the risk of cross-con-
lowest, most stable and most convenient storage tem- tamination during their storage and use in the lab-
perature, but vapour phase storage is generally con- oratory (see Principle 2).
sidered to be safer (see Appendix 1). Electrical
storage systems provide a very practical and mainte-
nance-free, low temperature storage solution. Principle 2: Assurance of the quality
However, in a multi-user environment, such systems of all materials and methods, and
are prone to the effects of temperature cycling in of their use and application, in
stored material, and in the absence of liquid nitrogen order to maintain the integrity,
or carbon dioxide back-up systems, they are at high validity, and reproducibility of any
risk in the event of loss of power supply.
work conducted
The failure of liquid nitrogen refilling procedures
can result in the loss of valuable cells and tissues, so The aim of quality assurance is to confirm the consis-
it is vital that there are effective training and mon- tency, traceability and reproducibility of in vitro cell
itoring procedures for the filling and maintenance and tissue work. Each laboratory should have desig-
of liquid nitrogen containers. In addition, it is advis- nated persons to oversee the quality assurance of:
able to store aliquots of important stocks at more
than one storage site. — the cells and tissues;
— growth media and all other materials;
— the methods, protocols, and SOPs;
1.5 Microbial, Viral and Cellular Cross- — the equipment and its maintenance;
contamination — the recording procedures; and
— the expression of results.
Contamination with bacteria, yeast and other fungi
can result in the complete loss of cultures. Undetected
contamination with slow growing micro-organisms, or 2.1 Cells and Tissues
with micro-organisms resistant to antibiotics, can
have a significant impact on the quality and/or valid- A laboratory should have specific protocols or SOPs
ity of data obtained from in vitro systems. The most for the receipt of new or incoming cells and tissues,
common example of such an infection is mycoplasma. and for the handling, maintenance and storage of
There are various potential sources of viral contam- all cells and tissues, with regular monitoring for
ination, including the operator, cell culture reagents of compliance. The following are among the factors to
animal origin, and cells or tissues of animal origin. All be considered:
cell and tissue culture facilities should therefore have
appropriate measures for minimising the risk of micro- — authenticity;
bial and viral infections and for their detection. — morphological appearance;
Viruses can cause lytic infections, thus destroying — viability;
the host cells, but may also become established as per- — growth rate;
sistent, sub-lethal infections, which are maintained — passage number and/or population doublings;
with passage of the host cell line. Many cell lines both — functionality;
carry and express virus sequences without producing — differentiation state;
infectious virus particles. In a small number of cases, — performance controls specific to the application;
infectious human pathogens are released into the cul- and
ture medium from lymphoblastoid cell lines (for exam- — contamination and cross-contamination.
2.2 Other Materials and In Vitro Culture glassware); and lack of toxicity (for example, plas-
Conditions tic, absence of detergents, and rubber components).
Appropriate procedures are necessary for the
The quality control of media, supplements and purchase, installation, commissioning, correct use,
additives is both time-consuming and expensive. performance monitoring (for example, calibration)
Since most of these materials are obtained commer- and maintenance of the following:
cially, the supplier should be expected to operate
according to standards appropriate to their supply — low temperature storage refrigerators;
and use, and to provide the relevant quality control — incubators;
documentation (Table 1). — laminar air flow and safety cabinets (see
The user laboratory has the responsibility: Appendix 2), and other sterile work areas;
— automatic pipettes and pipettors;
— to confirm that all the materials to be used are — sterilisation ovens and autoclaves; and
suitable for their intended purposes; — analytical and production equipment.
— to ensure that all materials are appropriately
handled, stored and used; and European Norms and ISO standards can be adopted
— to monitor batches of materials with regard to for these areas, and in some cases, compliance may
changes or variations which may affect their use be a legal requirement (for example, for pressurised
(for certain critical reagents, for example, gases, such as carbon dioxide/air for cell cultures,
serum, pre-use testing may be necessary). where there will be ISO standards for the gases, and
safety standards for the cylinders and pressure reg-
ulators).
In the case of critical reagents, the manufacturer
cannot be expected to know the user’s specific
requirements. The user should therefore define a
specification to include general details of the Principle 3: Documentation of the
reagent, such as quality controls for identity (com- information necessary to track the
position), purity and activity and stability. Where materials and methods used, to
relevant, the specification should include compli- permit the repetition of the work,
ance with international standards (such as ISO and to enable the target audience to
standards or pharmacopoeial protocols). understand and evaluate the work
All other working materials which come into
direct contact with cell and tissue cultures should In cell and tissue culture, as in any practical sci-
be regularly monitored, and appropriate procedures ence, clear documentation of the systems used and
should be in place for ensuring; the quality of cul- procedures followed is mandatory, in order to per-
ture vessels and surface coatings; the cleanliness mit the traceability, interpretation and repetition of
and sterility of any re-used equipment (for example, the work. Therefore, accurate records of cell type,
Table 1: Assessment of the quality of reagents used in cell and tissue culture
Quality assessor
Reagent Parameter Supplier End user
Serum Sterility and endotoxin testing +

Physical and biochemical analysis +
Functional testing + (general) + (specific)
Basal medium, complete medium Sterility testing +

(e.g. serum-free medium), additives Physical and biochemical analysis +
(e.g. non-essential amino acids) Functional testing + (general) + (specific)
Detachment solution (e.g. trypsin/EDTA) Sterility testing +

Functional testing + +
Surface coating for cell attachment Sterility +

Functional test + +
origin, authentication and characterisation, and of — type and origin of culture-ware (types and sup-
the materials used and the culture techniques per- pliers of flasks, Petri dishes, T-flasks, roller bot-
formed, are essential. tles, etc.);
The documentation should be retrievable, and — laminar air flow and safety cabinet testing, cali-
should include: bration, maintenance and repair;
— monitoring of humidity (if appropriate), temper-
— the objective of the work; ature and CO2 levels in incubators;
— the rationale for the choice of procedures and — monitoring of refrigerator and freezer tempera-
materials used; tures;
— the materials and equipment used; — monitoring of liquid nitrogen level and/or tem-
— the origin and characterisation of the cells perature in storage containers;
and/or tissues; — sterility controls (for example, autoclaving,
— the laboratory records, including results, raw sterility tests); and
data and quality control records; — regular maintenance and calibration of all other
— cell and tissue preservation and storage proce- critical apparatus (according to manufacturers’
dures; and manuals).
— the protocols and SOPs used, and any deviations
from them. The level of monitoring and testing may vary, from
installation of alarms for research and development
In some circumstances, for example, where compli- work, to continuous monitoring of calibrated moni-
ance with GLP or Good Manufacturing Practice toring systems for critical work.
(GMP) is required, there should be formal proce- With regard to the in vitro system, critical infor-
dures for the retrieval and review of documenta- mation must be recorded, to permit tracing of the
tion, and for resolving any questions or disputes history of the biological material, its characteris-
that may arise. tics, and the treatments, manipulations, measure-
ments and procedures applied to it, including
statistical procedures used to analyse the results
3.1 Origins of Cells and Tissues obtained.
Cell and tissue preservation and storage details
A minimal set of information is essential when should include (but not be limited to) the following
working with cells or tissues of animal or human (Table 3):
origin (Table 2).
— type of cell or tissue, passage/identity number;
— cryoprotectant used, and its concentration;
3.2 Handling, Maintenance and Storage — number of cells and volume per cryovial;
— position in storage container;
It is essential that records should be kept on the fol-
— viability and plating efficiency after thawing;
lowing:
and
— date and operator.
— culture media (including all supplements and
additives) and other solutions and reagents
Any changes in storage location should be formally
(including details of supplier, batch, storage
recorded and, when appropriate, relevant notifica-
requirements, expiry date), and methods of
tion should be given (for example, to the owner,
preparation (these may be generically specified
safety officer or quality control personnel).
in SOPs for research and development work, but
The disposal procedures for culture laboratory
for specific standards, the traceability of each
waste (used solutions, toxic treatments, biological
procedure to ensure the use of appropriate
reagents may be required); materials, etc.) must be documented, and compli-
ance with them should be ensured.
— culture substrate (type and supplier of coating
material, for example, collagen, fibronectin,
laminin, poly-D-lysine, Matrigel®, basal mem- 3.3 Reporting
brane), and recording of the coating procedures,
where applicable; and Effective communication is an essential part of cell
and tissue culture work, so careful attention should
— procedures for preparation or use of cells or tis- be given to the reporting procedures used.
sues. The format of a report will depend on the target
audience, for example, in-house personnel, a client
The records on handling, maintenance and storage or sponsor, a regulatory body, the scientific com-
related to culture-ware and equipment should munity, or the general public. The person(s)
include: responsible for the report should be identified.
Table 2: Examples of requirements for documentation concerning the origins of cells and
tissues
Isolated organs and Primary cultures

tissues of animal of animal All materials of
origin (e.g. rat origin (e.g. rat human origin Cell lines
brain tissue) hepatocytes) (e.g. cord blood) (e.g. Balb/c, 3T3)
Ethical and safety + + + Applicable, if human or

issues involving recombinant
DNA or pathogens
Species/strain + + + +
Source + + + +
Sex + + + +
Age + + + +
Number of donors + + If applicable na

Health status + + + +
Any special + + + +
pre-treatment
Organ/tissue of origin + + + +
Cell type(s) isolated + + + +
Isolation technique + + + +
Date of isolation + + + +
Operator + + + +
Supplier + + + +
Informed consent na na + If human, may be
applicable
Material transfer na na + +
agreement
Medical history na na + (if available) If human, may be
of donor applicable (if available)
Pathogen testing If applicablea If applicablea +a +a
Shipping conditions + + + +
State of material + + + +
on arrival
Cell line identification na na na +

and authentication
Mycoplasma testing na nab nab +
aScreening tests for animal colonies or donors of cells and tissue may be appropriate.
bMay be important if material is preserved for longer term use (e.g. as feeder layers for other cultures).
na = not applicable.
Where appropriate, the report should be formally and a discussion of the outcome. It should also be
authorised for its intended purpose. made clear that the whole study was established
A high-quality scientific report should cover the and performed in accordance with any relevant
objective of the work, the protocols and SOPs standards, regulations, statutes, guidelines or
used, planning and experimental design, the exe- guidance documents, and safety and quality
cution of the study, data collection and analysis assurance procedures.
Table 3: Examples of requirements for documentation concerning the handling,

maintenance and storage of cells and tissues
Isolated organs and Primary cultures

tissues of animal of animal All materials of
origin (e.g. rat origin (e.g. rat human origin Cell lines
brain tissue) hepatocytes) (e.g. cord blood) (e.g. Balb/c, 3T3)
Ethical and safety na na + may be applicable, if

issues human or involving
recombinant DNA or
pathogens
Morphology + + + +
Histopathology + na If applicable na
Quarantinea na + + +
Purity of isolation + + + +
Phenotype na + If applicable +
State of differentiation na + + +
Type of cultureb + + + +
Culture mediumc + + + +
Feeding cycles + + + +
Growth and survival + + + +

characteristicsd
Initial passage na na + +
number
Confluency at na na + +
subculture
Subculturing detailse na na + +
Induction of na + + +
differentiation
Identification and +f +f + +
authentication
Ageingg + + + +
Mycoplasma testing If applicableh If applicableh If applicableh +
aisolation from other cultures; btype of culture (e.g. monolayer, organotypic, suspension culture); ctype of culture
medium, additives and supplements and volumes used; dgrowth and survival characteristics (e.g. cell survival, time of
cell maturation, expression of cell-specific markers, ageing, initial density at plating, doubling time); esubculturing
details (e.g. date of sub-culture, subculture intervals, split ratios; seeding densities, perfusion rate); fcells and tissues
should be traceable to a particular animal or set of animals; greplication limits, passage number/population doublings
for the cells and/or maximum passage number; hwhere there may be a potential risk to other work or where risk
assessment of original tissue shows high risk of infection.
When submitting a report on cell and tissue Principle 4: Establishment and

culture work, a minimum set of information maintenance of adequate measures
should be included, which covers the origins of to protect individuals and the
the cells, the characterisation, maintenance and environment from any potential
handling of the cells, and the procedures used hazards
(see Tables 4 and 5). A statement of compliance
with the GCCP principles should also be National and local laws, based on moral and ethical
included. principles, govern safety in the workplace in most
Table 4: Details to be included in papers for publication in journals, using the example of
mouse 3T3 cells
Details Supplier details
Type of culture Continuous cell line na

Cell/tissue type Fibroblast-like na
Species Mouse na
Origin Balb/c3 embryo na
Description 3T3 na
Catalogue/product number ATCC 407/351C ATCC, Mannassas, VA, USA

Clone A31 86110401
Basic culture medium Ham’s F12 Gibco, Paisley, UK
Serum 10% newborn calf serum (NBCS) Gibco
Antibiotics 100U/ml penicillin, 100µg/ml streptomycin Gibco
0.25µg/ml Fungizone
Other additives 4mM glutamine ICN-flow, Irvine, UK
Complete medium No further comment na

Frequency of medium change At subculture, when used na
Culture flasks for stock cells 24cm2 angle-necked tissue culture flasks Nunclon, Roskilde, Denmark,
(163371) or 80cm2 filter closed flasks (167008) or Scientific Laboratory
Supplies, Nottingham, UK
Culture plates for test 96-well tissue culture plates (167008) Nunclon
Culture well inserts Not used na
Surface coating Not used na

Subculture frequency At confluency na
Subculture split ratio 1:6 na
Detachment solution 0.25% trypsin/EDTA Cambrex Bio Science,
Wokingham, Berks., UK
Usable passage range 25–45 na
Passage number at receipt 30 na

Passage number at use 35–40 na
Maintenance conditions 37°C, 5% CO2 in air na
Storage conditions Stock cells in liquid nitrogen in 40% NBCS/ na
20% DMSO
Use 3T3-NRU phototoxicity test na
Relevant Standard Operating OECD TG 427, EU B.29 na

Procedures/guidelines
References 12, 13 na
Further comments None na
countries. Many countries also issue guidelines on 4.1 Risk Assessment

occupational health and laboratory safety, and indi-
vidual laboratories may also have rules which reflect Identifying and evaluating risks, and taking appro-
local circumstances. Thus, the guidance on safety in priate action to avoid or minimise them, are the
the cell culture laboratory given here in no respect foundations on which safety is built. In the work
replaces these laws and regulations, but rather draws environment, and particularly in the laboratory,
attention to certain aspects of them and highlights where hazards may be complex and their evaluation
issues specific to the in vitro culture of animal and requires specialist knowledge, risk assessment
human cells and tissues. In many countries, each lab- should be performed in a structured way.
oratory is required to appoint a biological safety offi- Furthermore, the results of such risk assessments
cer, and this individual should be involved in the should be recorded, not only to confirm that they
safety evaluation of any cell culture procedures. have been carried out and appropriate action taken,
but also to act as a reference document for individ- medication or to a medical condition) should seek
uals performing the tasks assessed. These assess- expert medical advice before they are allowed to
ments should be reviewed at regular intervals, to work in a laboratory where cell and tissue culture is
take into account any changes in local practice, performed.
national or international regulations, or increases The safety conditions highlighted below relate
in scientific knowledge. not only to the safety of individual cell and tissue
It is important to pay particular attention to risks culture workers, but also to that of their colleagues,
which may be specific to, or more significant in, cer- the general public and the environment.
tain groups of workers. For example, where women Some of the areas of concern with regard to gen-
of reproductive age may carry a (possibly undiag- eral laboratory safety, and to which it might be
nosed) pregnancy and would be at greater risk from appropriate to apply risk assessment, are shown in
the effects of certain chemicals, such as teratogens Table 6. Hazards of particular concern in the cell or
or biological agents. Similarly, persons with a tissue culture laboratory are further discussed in
diminished immune response (for example, due to Sections 4.2 and 4.3, below.
Table 5: Details to be included in papers for publication in journals, using an example of

primary/early passage human cell culture
Details Supplier details
Type of culture Primary cell culture na

Cell/tissue type Keratinocyte na
Species Human na
Origin Foreskin QMC Hospital Trust, Nottingham, UK
Ethical permission Required Ethics Committee, QMC Hospital Trust
Supply to other users Not permitted

Transport solution Phosphate-buffered saline Gibco, Paisley, Scotland
Basic culture medium Epi-Life® Medium Cascade Biologics, Mansfield, Notts., UK
Serum None na
Antibiotics 100U/ml penicillin, 100µg/ml streptomycin Gibco
Other additives HKGS Kit (5-001 5) Cascade Biologics

Calcium chloride In-house
Complete medium No further comment na
Frequency of medium change Every 2 days and at subculture na
Culture flasks for 24cm2 tissue culture flasks (163371) Nunclon, Roskilde, Denmark, or Scientific
establishing cultures Laboratory Supplies, Nottingham, UK
Inserts Not used na
Surface coating Not used na

Subculture When 50–80% confluent (not when na
100% confluent)
Subculture split ratio 1:5 or 1:10 na
Detachment solution 0.25% trypsin/EDTA (R-001-100) with Cambrex Bio Science, Wokingham,
trypsin-neutralising solution (R002-100) Berkshire, UK
Usable passage range 1–4 na
Maintenance conditions 37°C, 5% CO2 in air na

Storage conditions Stock cells in liquid nitrogen, in 90% fetal na
calf serum/10% DMSO
Passage number at use 3 na
Culture plates for use 96-well plates (167008) Nunclon
Use 3T3-NRU phototoxicity test na
Relevant Standard Operating OECD TG 427, EU B.29 na

Procedures/guidelines
References 14, 15 na
Further comments None na
Once a risk assessment has been carried out, all pliers. For any substances which are potentially
relevant personnel must be made aware of the hazardous to health (for example, mutagens, cry-
potential hazards associated with their work, and oprotectants, labelling dyes), these data should
must be trained in the necessary precautions (typi- form the basis of a risk assessment for the use of
cal precautions are shown in Table 7) and desig- this chemical, as the level of risk will vary, depend-
nated safety procedures, as well as in the ing on, for example, the quantities being used and
appropriate use of the safety equipment required the techniques being employed. This is covered by
(including personal protective equipment) and the national legislation in some countries. Approved
appropriate handling of spills. waste disposal procedures should always be fol-
lowed.
Materials being tested in in vitro toxicity tests
4.2 Hazards Related to Cell and Tissue represent a particular problem, particularly if the
Culture Work study requires that they be anonymously coded and
supplied via an independent, external source.
Hazards can be categorised into three main groups: Although the concentrations used in the final test
physical hazards, chemical hazards, and biological solutions may be very low, the storage of the bulk
hazards. A risk assessment plan should consider all material and its handling can represent a signifi-
these hazards in relation to the proposed work. As cant potential risk. It should always be possible to
already mentioned, this assessment should not be break the code in the event of an accident.
limited only to the laboratory and laboratory per- Particular care should be taken with certain kinds
sonnel, but should also cover risks to people in the of materials, such as when women of reproductive
entire facility, people in the external environment, age may be exposed to teratogenic test materials
and to the environment itself. This is not only a during an in vitro reproductive toxicity study.
vital aspect of basic research and testing, but is par-
ticularly important when cultured cells and tissues
are used for diagnostic purposes or for producing Biological hazards
therapeutic products, or when the cells and tissues
themselves are used for therapeutic purposes. Many different issues related to potential biological
hazards must be considered and, in certain cases,
monitored and recorded in the cell and tissue cul-
Physical hazards ture laboratory.
Risk assessments should address issues that
The cell and tissue culture laboratory does not pose could arise from the species of origin (i.e. human
any specific physical hazards. However, laborato- and primate cells of highest risk, see reference 7),
ries and workspaces should always be kept clean the health status of the donor, the available data
and tidy, and free of material stored on the floor or from microbiological screening tests, and the cul-
anywhere where it can cause risk to other people. ture and storage history (8). Although not usually
Any equipment or apparatus used should meet dangerous to the user, cells and tissues have the
national safety guidelines. Equipment such as auto- potential to permit the replication of viruses poten-
claves and laminar flow or microbiological safety tially pathogenic to humans, and should therefore
cabinets should have a programme of maintenance be routinely treated as if they are a potential health
for safe use, usually carried out at a minimum fre- risk (Table 7).
quency of once a year. The correct operation of All cells and tissues new to the laboratory should
equipment should also be regularly checked. be handled under a strict quarantine procedure,
Procedures should be in place for ensuring the including suitable precautions to prevent the
safest possible use of equipment connected with spread of potential contamination, according to the
ultra-violet light, lasers, radioisotopes, liquid nitro- general guidance given in Table 7, with additional
gen (see Appendix 1) and pressurised gases. controls, as necessary (such as the use of separate
dedicated media and equipment, and work by dedi-
cated staff). Horizontal laminar flow cabinets
Chemical hazards should not be used when handling cells, as such cab-
inets are designed to protect only the work area and
The cell and tissue culture laboratory is not a par- the air flow is directed toward the user.
ticularly dangerous place to work with regard to Where the nature of the work means that there is
chemical hazards. However, some chemicals have a significant risk of biological hazard, special pre-
ill-defined or unknown biological effects, so general cautions must be taken in accordance with national
safety standards should always be maintained to requirements, most of which, where infectious
protect workers against these uncertain hazards. organisms are concerned, are based on the World
Material Safety Data Sheets for all chemicals used Health Organisation classification for human
in the laboratory should be requested from the sup- pathogens (Appendix 3).
Table 6: Some areas of concern in general laboratory safety to which risk assessment should
be applied
Facilities (such as laboratories, offices, storage and sanitation): for example, are they appropriate and adequate for the
intended use, well maintained, and properly heated, ventilated and lit?
Security: depending on the work, are special security precautions required, (for example, for restricted access to
site/laboratories, and for removal of hazardous material from the site)?
Health and safety of staff: is the health and safety monitoring of staff regularly carried out and documented?
Laboratory equipment: is the equipment used certified as sufficiently safe for its specific and intended purpose?
Infectious/biohazardous materials: are hazard classification, receipt, processing, containment, storage and disposal
conducted correctly, with use of the appropriate protective equipment, clothing and other precautions?
Chemicals and radioactive substances: are the receipt, handling, storage and disposal of hazardous materials (for
example, radioisotopes, toxic compounds, flammable liquids) conducted according to the correct procedures?
Hazard prevention: are appropriate hazard prevention plans established, are staff regularly trained in these
procedures (for example, fire evacuations), and are they applied correctly?
Waste disposal: is a waste management procedure established that ensures prompt and safe removal from the clean
cell culture areas, followed by disposal according to approved procedures?
If the cells or tissues originate from a certified classification and control of this kind of work differs
source, such as a recognised cell bank, which pro- between countries, and countries may decide to
vides certification of freedom from certain contami- classify work at a higher or lower level when new
nants, this documentation may suffice for risk information on a particular vector/host system
assessment, provided that the cells have not been becomes available.
exposed to potential sources of contamination since Risk assessment is clearly a dynamic process, and
leaving the bank. However, it is recommended that, has to take into account new developments and the
as a minimum and where advisable, mycoplasma progress of science. It is the responsibility of the sci-
testing should be carried out on all samples entists involved to keep up to date with develop-
received. ments in this expanding field of activity, and at all
Due to the risk that the operators’ immune sys- times to respect national and international guide-
tems may not protect them against, for example, lines and requirements.
the tumorigenic growth of their own cells which
may have been altered via the in vitro procedures
(for example, by transformation, immortalisation, 4.3 Risk to the Environment
infection, or genetic modification), most national
guidelines make it unacceptable for operators to Risks to the environment are generally due to poor
culture cells or tissues derived from themselves or waste disposal, leading to contamination of water,
from other workers in the same laboratory, nor to air or soil, or the escape from containment of haz-
genetically manipulate such cells or tissues, or treat ardous materials. The environment can also be con-
them with potentially pathogenic organisms. taminated by release of biological material due to
Many countries have national safety committees, accidents, including transport accidents, and sys-
which establish guidelines for work with genetically tems should be put in place either to prevent or
modified organisms (GMOs) and help and require minimise the potential for such damage. Support
scientists to classify and perform their work at the from the local biological safety officer should be
appropriate biosafety level. Recombinant cells, (i.e. sought, if available.
those produced by genetic engineering or genetic
modification [terms used to cover most techniques
which artificially alter the genetic make-up of an Waste disposal
organism by mixing the nucleic acids of different
genes and/or species together]) will generally fall Methods of waste disposal appropriate to the work
within the requirements of such guidelines. The in hand must be identified during the risk assess-
Table 7: Typical precautions to be used to ensure operator safety when handling cells and
tissues
Hands should be washed or disinfected before and after handling cells.

An appropriate gown or laboratory coat should be worn, to be put on when entering the laboratory and removed when
leaving it.
Personal accessories (for example, rings, watches), which might compromise cell and tissue culture activities, should
be removed or covered up to prevent contamination.
If appropriate, gloves should be worn, and replaced immediately if torn or punctured or during extended work
sessions.
When handling cell and tissue cultures, workers must avoid transferring contamination on the hands from the culture
work to unprotected body parts (for example, eyes or mouth), clothing or items in the open laboratory environment.
As far as is reasonably practicable, all cell and tissue work should be performed in a Class II cabinet or other
appropriate (micro)biological safety cabinet (see Appendix 2). NB: certain cabinets, such as horizontal flow cabinets,
protect the cells and tissues, but not the user or the general environment.
Mouth-pipetting must be strictly prohibited.
All procedures should be undertaken by using methods that minimise the production of aerosols that might spread
contamination by micro-organisms or cells.
All disinfectants used should be effective and appropriate for the work.
All work surfaces should be cleaned with an appropriate disinfectant, before and after use.
The use of sharps should be avoided as far as is possible. Any used sharps should be disposed of safely according to
approved procedures.
All cultures should be clearly and unambiguously labelled.
ment process. These methods must protect not only before leaving the laboratory, or should be
the individual tissue culture workers themselves, placed in rigid, leak-proof containers before
but also their colleagues, the wider population, and being transported elsewhere for autoclaving or
the environment. Work with known pathogens and incineration.
GMOs must be performed according to the relevant
regulations (see above), including methods of waste
disposal. Where methods are not specified in these Transport
regulations, there is a requirement to assess and
justify all proposed methods of waste disposal as The transportation of any biological materials,
part of the risk assessment. Similarly, the appropri- chemicals (including liquid nitrogen) or other mate-
ate method of disposal of hazardous chemicals must rials (for example, dry ice) of potential risk to
be identified before work with them is undertaken. humans, animals, plants and/or the environment,
In line with the above precautionary principle, the must comply with national or international regula-
following minimum precautions should be taken tions (see, for example, http://www.iata.org/
when disposing of waste from the cell culture labo- whatwedo/dangerous_goods). They should be
ratory: packed so as to prevent spills in the case of break-
age, be correctly labelled (with appropriate hazard
— all liquid waste, with the exception of sterile symbols), and have the appropriate accompanying
media or solutions, should be either chemically documentation (materials safety data sheet, import
inactivated (by using sodium hypochlorite or form, export form, and CITES permit, if applica-
another disinfectant) or autoclaved before dis- ble). A typical materials safety data sheet for a cell
posal; and line is shown in Table 8. Where appropriate, the
International Air Transport Association (IATA)
— all solid waste contaminated with tissue culture guidelines should be followed, as they are stringent
liquid and/or cells should either be autoclaved and are recognised internationally (for regular
Table 8: Typical material safety data sheets for animal cell cultures (Containment Level 1 or
2), but without references to specific national and local legislationa
Cultures are not specifically defined as hazardous, but as live cells, they are potential biohazards, and should be
treated as if biohazardous.
Emergency Telephone Number:
To be used only in the event of an emergency involving a spill, leak, fire, exposure or accident.
Description:
Either frozen or growing cells shipped in liquid cell culture medium (a mixture of components that may include, but is
not limited to: inorganic salts, vitamins, amino acids, carbohydrates and other nutrients dissolved in water).
SECTION I
Hazardous Ingredients:
Frozen cultures may contain 5–10% dimethyl sulphoxide (DMSO).
SECTION II
Physical data:
Pink or red aqueous liquid.
SECTION III
Health hazards:
For Biosafety Level 1 Cell Linesb
This cell line is not known to harbour an agent known to cause disease in healthy adult humans. This cell line has
NOT been screened for Hepatitis B, human immunodeficiency viruses or other adventitious agents. Handle as a
potentially biohazardous material under at least Biosafety Level 1 containment.
This cell line is known to contain an agent that requires handling at Biosafety Level 2 containment. Such agents have
been associated with human disease. This cell line has NOT been screened for Hepatitis B, human immunodeficiency
viruses or other adventitious agents. Cell lines derived from primate lymphoid tissue may fall under the regulations
relating to blood-borne pathogens.
SECTION IV
Fire and explosion:
Not applicable.
SECTION V
Reactivity data:
Stable. Hazardous polymerisation will not occur.
SECTION VI
Method of disposal:
Spill: Contain the spill and decontaminate by using suitable disinfectants, such as chlorine bleach or 70% ethyl alcohol
or isopropyl alcohol.
Waste disposal: Dispose of cultures and exposed materials by autoclaving at 121°C for 20 minutes.
Follow all national and local regulations.
SECTION VII
Special protection information:
Handle as a potentially biohazardous material under at least Biosafety Level 1 containment. Cell lines derived from
primate lymphoid tissue may fall under regulations relating to blood-borne pathogens.
Handle as a potentially biohazardous material under at least Biosafety Level 2 containment. Cell lines derived from
primate lymphoid tissue may fall under regulations relating to blood-borne pathogens.
SECTION VIII
Special precautions or comments:
Recommended that appropriate safety procedures be used when handling all cell lines, especially those derived from
human or other primate material. For detailed discussions of laboratory safety procedures see references 15–18.
aThis generalised example of a material safety data sheet is based on one that can be found at
http://www.atcc.org/pdf/msds_animal.pdf; bBiosafety Levels 1 and 2 are broadly equivalent to European Containment
Levels 1 and 2.
updates, see www.wfcc.info). Before arranging international requirements. Some countries have, or
transport, the various legal requirements for export are preparing, legislation or regulations to control spe-
and import into the recipient country should be cific areas, such as the use of material of human ori-
considered, including ethical issues (such as the use gin. New controls are also being drafted in response to
of human cells or tissues of embryonic origin), dis- the challenges and opportunities presented by trans-
ease transmission, endangered species regulations plantation, regenerative medicine, stem cell research
(www.cites.org/), and bioterrorism regulations (see and GMOs. Ownership of cell lines and patents must
http://www.bt.cdc.gov/). also be dealt with appropriately, and special condi-
A cell culture may fall into any one of the classes tions may apply where cell cultures are involved (see
of biological material used for shipping purposes, reference 9). In addition, there are international
namely: agreements relating to the provision of organisms and
cell cultures that may be used for bioterrorism.
— diagnostic specimens;
— infectious specimens;
— biological products; or 5.2 The Use of Animal Material
— GMOs.
In general, any work involving animal material
should be in compliance with local and national leg-
Principle 5: Compliance with relevant islation on animal experimentation and the Three
laws and regulations, and with Rs (reduction, refinement and replacement) princi-
ethical principles ples of Russell & Burch (10). In addition, other eth-
ical issues may arise in certain circumstances.
From an ethical and legal point of view, it is desir- Examples include the use of cells derived from
able that high standards for cell and tissue culture endangered species (http://www.cites.org/), the pro-
should be established and maintained worldwide, so duction of monoclonal antibodies by the ascites
that accountability, safety and ethical acceptability method (see References: Monoclonal antibodies and
can be universally guaranteed, as far as is reason- ethics), and the pretreatment of animals with
ably practicable. The ethical and associated legal chemical inducers to provide cells for culture with
issues raised are extremely complex and beyond the specific biochemical properties (for example, hepa-
scope of these GCCP guidelines. However, all con- tocytes with elevated CYP450 enzyme levels).
cerned should maintain a sufficient level of aware- In order to minimise pain and distress, donor ani-
ness of the ethical issues related to cell and tissue mals should be handled according to the appropriate
culture work, and of public opinion and the relevant and approved procedures. As fetuses of many mam-
legislation at the national and international levels. malian species can already feel pain long before birth
At present there are no ethical guidelines relating (11), they should also be treated with the utmost care,
specifically to general cell culture practices, but var- again according to appropriate procedures.
ious guidelines, regulations and laws are in place Serum, and especially fetal bovine serum, is a com-
for dealing with cells and tissues of specific origin monly-used component of animal cell culture media.
and/or use. It is harvested from bovine fetuses taken from preg-
Before any studies are initiated, matters of ethi- nant cows during slaughter. Here again, the current
cal significance must be carefully considered. These practice of fetal blood harvesting poses ethical prob-
can be subdivided, from a GCCP point of view, into lems (blood is usually taken via cardiac puncture,
general ethical considerations and more-specific without any form of anaesthetic; 11, 12). Efforts are
considerations. being made to reduce the use of animal serum and,
From a general perspective, diligence in legal and where possible, to replace it with synthetic alterna-
ethical matters leads to data of higher value, since tives. A wide range of other cell culture materials
it can help to avoid waste of effort and can increase derived from animals (such as tissue extracts, extra-
confidence in the outcome of the study, to the ben- cellular matrix materials) also raise ethical concerns.
efit of all concerned, including the general public. Legal issues can also arise if animal-derived cells
The more-specific considerations include the eth- and tissues are found to be infected with viruses
ical implications of using material of animal and which could infect wildlife or species of agricultural
human origin, and GMOs. importance. For this reason, the discovery of such
viruses in cells and tissues may need to be notified to
the relevant authorities and appropriate action taken.
5.1 Laws and Regulations
At present, there are no international laws specifically 5.3 The Use of Human Material
governing cell and tissue culture practices. However,
any work involving animal or human pathogens has The use of human biological material is critical for
to be performed in compliance with national and medical research. It is particularly important that
researchers are aware of the need to handle such tions to be taken into account are similar to those
material in a responsible manner and in accordance involved in obtaining other human tissues, but it is
with local and national requirements. the use of the stem cells which requires effective
Those involved with the procurement, supply and regulation.
use of human biological material should maintain Before any human material is used for the estab-
proper records, to ensure appropriate traceability lishment of a new cell line, ethical approval should
and control of the applications of the material in be obtained from the relevant authority.
ways which are consistent with the nature of the
consent given by, or on behalf of, the donor. All use
of human tissue should be approved by the appro- 5.4 Genetically Modified Cells
priate ethics committee, and copies of such
approvals should be kept for reference. Where sam- The creation, storage, transport, use and disposal of
ples are provided to third parties, the custodian is genetically engineered cells are currently subject to
responsible for the safe keeping of the code which the requirements that apply to GMOs. This is a rap-
enables samples to be linked to individual donors, idly expanding field, and its long-term conse-
where appropriate and when necessary. quences are as yet unknown. It involves
Human material is usually procured either from manipulating genes and cells in ways that do not
specialised cell and tissue banks or from hospitals occur in nature, and for this reason, it raises sensi-
(13). Currently, most of the banks are run on a not- tive ethical issues. The above activities are regu-
for-profit basis. Nevertheless, some of them have lated in many countries, where, before any work is
been set up by private industries, particularly for initiated, relevant approval must be sought.
the production of engineered tissues. This raises
serious ethical concerns (including the transfer of
human material for profit), and has not yet been Principle 6: Provision of relevant and
dealt with adequately at the national level in most adequate education and training for
countries or internationally. In Europe, this area all personnel, to promote high
will be regulated under the EU Human Tissues quality work and safety
Directive (14).
Confidentiality with respect to the provision and The range of applications for cell culture is expand-
use of human tissue is governed both by law and by ing rapidly and involves an ever-broadening range
professional guidelines. A legal requirement in of technical manipulations (such as chemically
most countries is that, when dealing with human induced and genetic modifications) for use in basic
material, informed consent must be sought either and applied science, manufacturing, diagnosis, and
from the donor or from the donor’s family. efficacy and safety testing procedures, as well as for
Human tissue banks should be recognised as the providing therapeutic materials.
most legally and ethically acceptable approach to The competence of staff to perform their duties
the procurement and distribution of donated non- in a laboratory is central to ensuring that work is
transplantable human tissue for research, as they performed according to the standards of the organ-
are best equipped to deal with, and advise on, the isation in relation to its scientific, legal and safety
complex issues involved, including ethics, consent, requirements and obligations. This requires educ-
safety and logistics, as well as scientific questions. ation and training, as well as the regular monitor-
The removal of blood samples from human vol- ing of performance (Table 9).
unteers should only be performed by qualified per- A good basic education should be given in the
sonnel, and particular precautions should be nature and purposes of cell and tissue culture which
followed to minimise any risks. Such volunteers is an essential basis for any future training pro-
should also be considered to be donors, and docu- gramme. The basic principles of in vitro work, asep-
mented informed consent will be required. tic technique, cell and tissue handling, quality
The use of human embryonic stem cells involves assurance, and ethics should be included. It is also
serious ethical questions, because of their origins important that those working with material of ani-
and their potential uses. This is a relatively new mal or human origin should have a sufficient under-
research area, and, while some countries already standing of any additional laws or regulations that
have strict controls, other countries are currently will apply.
considering what laws and regulations should be Training should be seen as an ongoing process
introduced in the public interest. for improving and developing practical skills, and
The procurement of stem cells from early maintaining competence. Given its critical impor-
embryos and fetuses is a particularly sensitive tance to the success of any laboratory work, there
issue, because of the circumstances in which such should be a formally documented training pro-
embryos and fetuses become available. Stem cells gramme for all members of staff, including train-
can also be obtained from adult tissues and from ing records and regular reviews of training needs.
umbilical cord blood, where the ethical considera- To ensure the quality of work in the long term, it
Table 9: Culture techniques, procedures and regulations that should be included in a cell
culture laboratory training programme
Basic laboratory procedures

Understanding of the nature and purpose of SOPs
Microscopy
Centrifugation
Autoclave operation
Use and maintenance of laminar air flow or microbiological safety cabinets, incubators, cryostorage facilities
Maintenance of essential equipment
Laboratory design and safety
Risk assessment and risk management of in vitro work
Quality control
Waste disposal
Disinfection, fumigation and cleaning regimes
Basic culture procedures

Sterile technique and aseptic manipulation, including disinfection and sterilisation
The preparation, storage and monitoring of culture media
Cell and tissue culture isolation techniques
Cell viability testing and cell counting
Subculturing
Sterility or bioburden tests
Mycoplasma testing
Cryopreservation, storage and recovery of cells and tissues
Advanced and special culture procedures

Cell characterisation and authentication
Cell isolation and purification methods
Cell and tissue banking
Induction of differentiation
Complex culture techniques (for example, co-culture, culture on filter inserts, perfusion cultures)
Transfection and selection of stable cell lines
Use of bioreactors
Documentation and record keeping

General information and policies of the organisation responsible for the laboratory (operational issues, safety, quality
standards)
Laboratory data, equipment records, storage records
Occupational health and training records
Safety records
Quality assurance records, manuals and information
Laws and regulations

All laboratory staff should be made familiar with the institutional, national and international procedures, guidelines,
regulations and laws relevant to their work, such as the following:
— rules and policies of the organisation/institute
— allocation of responsibilities
— the containment of microorganisms;
— regulations on the use of animals and of animal cells and tissues; and
— regulations on the use of human cells and tissues.
is also important to link training with personal cedures, covering SOPs, general laboratory main-
development programmes for technical and scien- tenance, and safety and emergency procedures.
tific staff, in order to ensure they are progres- Training can be provided in-house by experi-
sively trained and educated in line with changing enced members of staff and/or visiting experts,
laboratory activities and demands. via accredited on-line programmes and/or
When new staff join a laboratory, their skills through attendance at external courses. For cer-
and experience should be assessed, and the need tain applications including product manufacture
for further training procedures in relation to and testing, and processing of cells and tissues for
their new jobs should be identified. These needs clinical use, training must be formally recorded
may include a variety of general and specific pro- and reviewed.
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assessment resource)
Web-links
http://www.hse.gov.uk/pubns/indg163.pdf (UK Health &
Safety Executive resource for general risk assessment)
Cell banks, cell line authentication and identification
http://www.who.org (World Health Organisation)
DSMZ – German Collection of Microorganisms and Cell http://www.osha-slc.gov/SLTC/laboratories/ (US
Cultures: Occupational Safety and Health Administration resource
http://www.dsmz.de for laboratory safety)
European Collection of Cell Cultures (ECACC): http:// http://www.bocgases.ie/saftey/othersaftey/pdf/cryogen-
www.ecacc.org.uk ics.pdf (guidance on the safe use of liquid nitrogen and
other liquid gases)
American Type Culture Collection (ATCC): http://www.
atcc.org http://www.iata.org/whatwedo/dangerous_goods
Appendix 1
Liquid Nitrogen
Work with liquid nitrogen probably poses the great- clothing or shoes, or spilled down the cuff of an
est single threat to the safety of cell culture work- insulated glove. Therefore, appropriate clothing
ers (as gauged by the number of individuals using it should always be worn (open-toed footware is not
and the potential severity of any accident), and for permissible, and clothing with loose cuffs, pockets
this reason is dealt with in greater detail here. and turn-ups should be avoided), with eye protec-
Details of general hazards, precautions, and first tion and insulated gloves (ideally, these should be
aid can be obtained from the suppliers of liquid loose-fitting for ease of removal, be made of imper-
nitrogen and of liquid nitrogen vessels (see, for meable material, and have close-fitting, elasticated
example, http://www.bocgases.ie/saftey/othersaftey/ cuffs).
pdf/cryogenics.pdf). Such relevant information The other hazard associated with liquid nitrogen
must be obtained, and its contents must be taken is that it can enter storage vials (due to inadequate
into account in the relevant risk assessments. A sealing) when they are immersed in the liquid
printed version should be placed in a readily acces- phase, and this may cause the vials to explode upon
sible location where it can be rapidly referred to, thawing. Therefore, steps must be taken to protect
before any work using liquid nitrogen is under- workers from the effects of such an explosion. As a
taken. minimum, workers must wear a full-face visor,
A serious hazard in the use of liquid nitrogen is insulated gloves and a long-sleeved laboratory coat
the risk of asphyxiation due to the displacement of when thawing vials from liquid nitrogen, and other
air by nitrogen gas within a confined area. Areas individuals must be kept clear of the immediate
where liquid nitrogen is stored or handled must area. The vessel containing the liquid in which the
therefore be well ventilated. In addition, oxygen- vial is being warmed, if judiciously chosen, can be
depletion monitors (wall-mounted and/or worn by used to further protect the worker by containing
staff), which can provide an early warning that the any flying debris and/or directing the force of the
level of oxygen is declining below a safe level, blast away from the worker.
should be used in areas where large numbers of
Such explosions could be particularly dangerous
storage vessels are held and/or significant amounts
if the vials contained pathogenic material.
of liquid nitrogen are handled.
Therefore, material known to be pathogenic must
Liquid nitrogen is frequently stored in pres-
not be stored in the liquid phase of liquid nitrogen,
surised vessels. Many countries have regulations
but instead should be stored in the vapour phase.
governing the design, construction, use, mainte-
Another reason for this is that transfer of patho-
nance, testing and other aspects of such pressurised
genic material between containers stored in the liq-
vessels (for example, the UK Pressure Systems
Safety Regulations 2000). In countries where no uid phase has been documented.1 Clearly, the
such regulations exist, similar precautions should greatest care must be taken to ensure that storage
be taken. In particular, cell culture workers should vessels containing pathogenic material are fully
ensure that they know how to operate such vessels sealed before placing them in storage, and that they
safely (see the user’s manual) and must have their will stay fully sealed under the intended storage
vessels maintained and tested on a regular basis. conditions.
Further useful information can be found at: 1Tedder, R.S. Zuckerman, M.A., Goldstone, A.H.,
http://www.hse.gov.uk/hid/land/comah/level3/5c9a7
Hawkins, A.E., Fielding, A., Briggs, E.M., Irwin, D., Blair,
bd.htm. S., Gorman, A.M., Patterson, K.G., Linch, D.C.,
Because of the ultra-low temperature of liquid Heptonstall, J. & Brink, N.S. (1995). Hepatitis B trans-
nitrogen (–196°C), it can cause severe frostbite to mission from contaminated cryopreservation tank.
exposed tissues, particularly if it is caught in loose Lancet 346, 137–40.
Appendix 2
Class II Biological Safety Cabinets
A Class II Biological Safety Cabinet (BSC) is — Ensure that a vessel of appropriate disinfectant
designed to perform two functions: a) to protect is on hand, in case of spillages.
the work materials (i.e. cell and tissue cultures in
this case); and b) to protect the worker from — Bear in mind that once the work has started, all
materials within the BSC are potentially con-
infectious agents that may be contained in the
taminated and should not be removed until after
work materials. Other cabinets (Class I and III) appropriate disinfection. This includes gloved
have been developed, each to serve only one of hands.
these functions 1, but, in addressing both require-
ments, the Class II cabinet is less robust if mis- — Do not subculture or otherwise manipulate more
used, and for safe and reliable performance of its than one cell or tissue culture system in the BSC
dual function, requires careful installation, main- at any one time. This is essential, to avoid mis-
labelling or cross-contamination.
tenance and practical use. Accordingly, the oper-
ators of BSCs require careful instruction as to — Use separate bottles of growth medium for each
their use. Supervisors must inform staff that cell or tissue culture system, as this will prevent
Class II BSCs are not substitutes for good aseptic the transfer of microbial agents between culture
technique; in particular, the airflows will not pro- systems or possible cross-contamination.
vide protection in cases of gross spillage, high
energy aerosols (for example, from centrifuges, or — Avoid rapid movements, which may interrupt
the air flow.
sprays) and physical disturbance. The following
are some typical precautions to help ensure the — When the work is completed, ensure that all
correct and safe use of a Class II BSC. materials and equipment are made safe. Place
all materials that need to leave the BSC in
— Before using the BSC, ensure that it is working appropriate transport containers, and disin-
correctly. Check the airflow indicators or the fect by either spraying or wiping. Disinfect the
negative pressure gauges. Most BSCs are fitted work area in case of spillage and splashes.
with alarms to indicate any unsafe operation
— Depending on the work being carried out, the
conditions. BSC may need to be decontaminated with
formaldehyde prior to further work being under-
— Use appropriate disinfection to decontaminate taken.1
surfaces before commencing work.
— Leave the BSC running for at least 10 minutes
— Ensure that all essential materials and equip- before switching it off, in order to remove any
aerosols generated during the work.
ment are placed in the BSCs before work is
started; this will reduce the risk of interruptions — Class II safety cabinets should be sited, installed
to the BSC air flow during use, and will reduce and commissioned according to national regula-
the risk of contamination. tions.
1 Jones,
B.P.C. (1998). Laboratory practice. In Safety Cell
— Do not place too many items in the BSC at any
and Tissue Culture (ed. G. Stacey, A. Doyle & P.
one time, as cluttering the work area may affect Hambleton), pp. 64–86. Dordrecht, The Netherlands:
the air flow. Kluwer Academic Publishers.
Appendix 3
Classification of Infective Microorganisms by WHO Risk Group1
Risk Group 1 Risk Group 3

(no or low individual and community risk) (high individual risk, low community risk)
A microorganism that is unlikely to cause human or A pathogen that usually causes serious human or
animal disease. animal disease, but does not ordinarily spread from
one infected individual to another. Effective treat-
ment and preventive measures are available.
Risk Group 2
(moderate individual risk, low community risk)
Risk Group 4
A pathogen that can cause human or animal dis- (high individual and community risk)
ease, but is unlikely to be a serious hazard to labo-
ratory workers, the community, livestock or the A pathogen that usually causes serious human or
environment. Laboratory exposures may cause seri- animal disease and that can be readily transmitted
ous infection, but effective treatment and preven- from one individual to another, directly or indi-
tive measures are available, and the risk of spread rectly. Effective treatment and preventive meas-
of infection is limited. ures are not usually available.
1World Health Organisation. (2004). Laboratory Biosafety Manual, 3rd edn, p. 1. Geneva, Switzerland: WHO. Website
http.//www.who.int/csr/resources/publications/biosafety/Biosafety7.pdf (Accessed 8.06.2005)
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Good cell culture practices

Article in In Vitro Cellular & Developmental Biology - Animal · March 2004

Sandra Coecke Michael Balls

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John Davis Gerhard Gstraunthaler

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Guidance on Good Cell Culture Practice

A Report of the Second ECVAM Task Force on Good Cell Culture

Isolated organs or tissues

— exposure of normal cells and tissues to irradia-

Reagent Parameter Supplier End user

Serum Sterility and endotoxin testing +

Basal medium, complete medium Sterility testing +

Detachment solution (e.g. trypsin/EDTA) Sterility testing +

Surface coating for cell attachment Sterility +

Isolated organs and Primary cultures

Ethical and safety + + + Applicable, if human or

Number of donors + + If applicable na

Cell line identification na na na +

Table 3: Examples of requirements for documentation concerning the handling,

Isolated organs and Primary cultures

Ethical and safety na na + may be applicable, if

Growth and survival + + + +

When submitting a report on cell and tissue Principle 4: Establishment and

Details Supplier details

Type of culture Continuous cell line na

Catalogue/product number ATCC 407/351C ATCC, Mannassas, VA, USA

Complete medium No further comment na

Surface coating Not used na

Passage number at receipt 30 na

Relevant Standard Operating OECD TG 427, EU B.29 na

countries. Many countries also issue guidelines on 4.1 Risk Assessment

Table 5: Details to be included in papers for publication in journals, using an example of

Details Supplier details

Type of culture Primary cell culture na

Supply to other users Not permitted

Other additives HKGS Kit (5-001 5) Cascade Biologics

Surface coating Not used na

Maintenance conditions 37°C, 5% CO2 in air na

Relevant Standard Operating OECD TG 427, EU B.29 na

Hands should be washed or disinfected before and after handling cells.

Mouth-pipetting must be strictly prohibited.

All cultures should be clearly and unambiguously labelled.

Basic laboratory procedures

Basic culture procedures

Advanced and special culture procedures

Documentation and record keeping

Laws and regulations

References tion, procurement, testing, processing, preservation,

1Copies available on request from NSW Agriculture Animal Welfare Unit.

Class II Biological Safety Cabinets

Classification of Infective Microorganisms by WHO Risk Group1

Risk Group 1 Risk Group 3

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