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BioSafety Journal
Pontificia Universidad Católica de Valparaíso
ISSN: 1366 0233
Vol. 1, Num. 1, 1995
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BioSafety
Volume 1, Paper 8 (BY95008), June 27th 1995
Online Journal, URL - http://bioline.bdt.org.br/by
Risk assessment of animal cell culture procedures
G. Stacey
Centre for Applied Microbiology and Research, Salisbury,
Wiltshire, SP4 OJG, UK.
E-mail: ecacc@ecacc.demon.co.uk
Received June 1st 1995
Code Number: BY95008
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INTRODUCTION
The generation of continuous cell lines from animal and human
tissues has yielded valuable tools for biological studies.
Some of these cell lines have also proved important hosts for
expression of recombinant DNA. The fact that cell culture is
used in diverse disciplines has meant that to a large extent
it has evaded specific attention regarding risk assessment.
The primary concern relating to safety in cell culture is that
animal cells can provide a suitable medium in which
microorganisms, notably viruses, can multiply. There are a
number of levels at which the culture and manipulation of
animal cells should be considered from the point of view of
safe handling. This overview attempts to outline the type of
approach which should be adopted in risk assessment along with
some practical suggestions to promote safe handling of cell
cultures.
RISK ASSESSMENT
When using any new procedure or modifying an existing protocol
it is essential to carry out a risk assessment of all aspects
of the work process, namely starting materials, culture
procedures, product purification and waste disposal. For most
chemicals and reagents used in cell culture there are standard
texts and sources of information, most obviously the
manufacturer, which enable rapid assessment of risk based on
the properties of the reagent, its physical form, the
quantities used and the procedures to which it is to be
subjected. However, there are a number of factors unique to
the manipulation and culture of animal cells which make risk
assessment a more difficult and sometimes uncertain process.
The following paragraphs aim to identify the safety concerns
specifically relating to cell culture and to give some general
guidance for risk assessment in these areas.
(1) Undefined Components of Growth Media.
Numerous growth medium supplements, notably foetal bovine
serum, can provide a potential source of virus contamination
(Erickson et al. 1991). In general such supplements
cannot be readily sterilised since this often results in their
inactivation or degradation. Thus, it is important to obtain
such materials from suppliers which guarantee that they stock
only from uninfected sources or accredited virus free animal
herds. Some manufacturers may carry out tests for the
detection of adventitious agents, but this is limited to
specific organisms (eg. mycoplasma and Bovine Viral Diarrhoea
Virus). Obviously, if human (and primate) sources of undefined
reagents are avoided then the risk of infection in laboratory
workers by this route is minimised. There are a number of
serum free defined culture media and sources of recombinant
growth factors which eliminate the possibility of virus
transmission via culture media components. Unfortunately,
these are not yet widely used and they can be very expensive.
However, the search for a cheaper culture medium may lead to
the use of less pure reagents subjected to lower standards of
quality assurance. Thus, in the long term, the use of less
expensive culture media may be counterproductive both in terms
of safety and quality.
2) Cells and Adventitious Agents
Risk assessment of animal cell cultures is a potentially
confusing area since the cells are essentially undefinable and
prone to variation. However, the primary cause for concern,
in relation to laboratory work with cells, is the potential of
cell cultures to sustain virus or other organisms which might
infect laboratory workers. Thus practical approaches to risk
assessment of animal cell cultures have been based on the
virological risk represented by the species and tissue of
origin (Frommer et al., 1993). Human and primate cells
derived from blood and lymphoid tissue are of greatest concern
as carriers of serious human pathogens. Whilst non-human,
non-primate sources of cells in general represent a much lower
risk to laboratory workers some rodent viruses can cause human
infection (eg. Hay, 1991; Mahy et al., 1991).
Many cell lines undergo multiple passages in different
laboratories. The history of such lines is rarely recorded and
thus the risk of cross-infection with infectious agents from
other cell lines and contaminated reagents cannot be assessed.
For primary cells isolated directly from tissue the risk of
infection is related directly to contamination from the tissue
of origin (ie: microbiological status of the animal or colony
of origin) and the culture medium used. Probably the most
likely organisms to pass between cell lines and establish an
infection are mycoplasmas and acholeplasmas. These organisms
survive well in the environment and unfortunately are
commonplace in animal cell cultures and can be extremely
difficult to eradicate. Some of the most common contaminant
species in cell cultures (eg Mycoplasma orale, M. salivarum,
M. hominus, M. fermentans) probably originate from the
commensal flora of laboratory workers and feotal bovine serum
(Del Giudice and Gardella, 1984). While mycoplasma infection
can have drastic effects on cells, the species identified in
cell culture are not generally associated with human disease
except for M. pneumoniae. Thus, although the presence of
adventitious agents can have serious consequences for infected
cell lines, they are unlikely to represent a serious health
threat to laboratory workers using good aseptic technique.
A more insidious problem, is the potential for cell lines to
harbour and secrete virus while showing no overt signs of
infection. This unrecognised factor and the need to prevent
the spread of contaminants between cultures are the primary
indicators for care in cell lines containment (see below).
The situation is simplified (in terms of risk assessment only)
when a hazardous virus is used to infect cells. The culture as
a whole then assumes the higher hazard level for the virus.
Published literature and the experience of other workers
regarding a particular cell culture of interest may also be
valuable in risk assessment. However, direct transcription of
specific data (eg virus testing) should be supported by proof
of the common origin of cells in use and those in the
literature utilized in risk assessment.
3) Cell products
Cell products naturally released from animal cell cultures do
not usually represent a hazard. Generally, this is a problem
relating to direct exposure to purified products which can be
alleviated if direct exposure of the body surfaces and
respiratory tracts of laboratory workers is minimised. Cell
products produced at high concentration, for example from some
recombinant organisms, demand a higher priority in risk
assessments and should be assessed for their toxic properties,
persistence in the environment and their ability to cause
adverse immune reactions. The potential for exposure to
transforming proteins, such as E1A adenovirus protein and SV4
T antigen, is also an important consideration. It should be
borne in mind that scale-up procedures that are aimed to
produce high and concentrated yields of product may represent
significant hazards which were of lesser consequence at
research and development stages. Control of products released
inadvertently into the laboratory environment should be
provided for by adequate cleaning and decontamination
procedures. This is important both as a routine procedure as
well as for the containment of spillages. Together, these
approaches will prevent the build up of any cell culture
product in the working environment.
4) Cell processing procedures
Once all the various components of a particular process have
been assessed individually it is important to review the
proposed physical processes to be used and assess the level of
containment required at each stage. Any procedures where
aerosols are generated or materials may be accidentally
transferred directly to an operator's tissues and/or blood
stream (eg use of hypodermic needles) should be reconsidered.
In such cases, it is important to identify alternative
procedures or, if this is not possible, to ensure that
reasonable precautions are taken to contain aerosols
adequately and protect the operator. In procedures whereby
cells are lysed aggressively it will be necessary to consider
not only the proteins but also the DNA (recombinant or
genomic) which is being released. Whilst the hazards of
non-viral naked DNA are as yet not clearly quantified it is
prudent to limit its spread.
5) Decontamination procedures
It is important that the decontamination procedures are chosen
specifically for each process. Selected disinfectants should
be checked for their efficacy against those microorganisms
likely to be present and the use of mixtures of disinfectants
and/or cleaning agents should be checked for their
compatibility. Decontamination procedures should be recorded
as laboratory protocols with instructions for the preparation,
use and regular replacement of `in use dilutions' of
disinfectants and cleaning agents. Cell culture contaminated
materials should be treated to ensure that they cannot carry
any infectious particles when leaving the laboratory. In the
case of materials likely to be heavily contaminated this is
readily achieved by destructive autoclaving (ie in an
atmosphere of pure steam at 134C and 1 atm pressure for 30
mins). The mechanism of waste disposal should also be
assessed to prevent outgoing waste contaminating new reagents
and to prevent build up of waste which in addition may then
become a secondary source of contamination within the
laboratory.
PRACTICAL APPROACHES TO SAFE HANDLING OF ANIMAL
CELLS
1) Use of Safety Cabinets.
As discussed above, cell lines may carry undetected
microorganisms. Therefore all cell cultures should be handled
within an appropriate microbiological safety cabinet. Where no
human pathogen exceeding category 2 (see below) has been
identified a Class II cabinet may be used for the purpose of
containment. It is vital that such equipment is installed,
monitored and maintained correctly (eg as given in the British
Standard BS5726 (2)). In addition all staff involved in
tissue and cell culture work should receive training in the
correct use of safety cabinets.
2) Organisation of work
Work practices should be designed to ensure that infected and
uncharacterised cultures do not contaminate culture media and
other "clean" (characterised) cultures. This can be readily
achieved where there is the facility for separate areas for
media preparation, "clean" cell cultures and infected or
uncharacterised cells. Where this is not the case work can be
organised into sessions of increasing risk of contamination
through each day followed by stringent decontamination. Such
approaches, while important for safety, are also of scientific
benefit in preventing the transmission of agents, which may
lead to altered cell characteristics, between cell lines.
This chronological form of quarantine is absolutely dependent
on a high level of training in aseptic technique amongst the
laboratory staff (see below).
3) Characterisation of Cell Cultures.
In order for risk assessment of a cell culture procedure to be
accurate, it is important to confirm the authenticity of the
cells in use. When dealing with cell lines with unique
characteristics or primary cells isolated directly from tissue
there is little chance that the wrong cells might be used.
However, for cell lines obtained from another laboratory there
may be no proof of identity. Thus, it is necessary at least
to have evidence of the species of origin of a cell line and a
number of techniques are available which have been used for
confirming the identity of cell cultures (Stacey et al
1992). It is also important to realise that the same cell
line obtained from different sources can show phenotypic
variation due to different culture histories.
4) Training
Probably the most important protection that the laboratory
worker has against infection is aseptic technique. This
prevents transmission of contaminated particles between the
manipulation procedure and the environment (which includes the
operator). Thus, while primarily intended to protect research
material from contamination, aseptic technique is an important
element in containment of infectious organisms. It is vital
that all laboratory staff should receive training in aseptic
technique which will benefit the productivity and quality of
their work as well as their safety. Staff should also be
trained in the correct practices for disinfection,
sterilisation (ie autoclaving) and fumigation which provides a
secondary level of containment. Further training in the
manipulation of special hazards (eg cytotoxic drugs, oncogenic
materials, genetically modified organisms) should also be
introduced as required.
NEW DIRECTIVES ON SAFETY AND BIOLOGICAL AGENTS
Within Europe, a directive from the European Commission
(90/679/EEC) has lead to a unified classification of
biological agents. This has been prepared with requirements
and recommendations for containment of each class of agent.
This classification of biological agents is based on those
used in a number of member states of the European Union, for
example that of the UK Advisory Committee on Dangerous
Pathogens (ACDP, (1)), as are the requirements for containment
of pathogens. As undefined complex biological systems, which
may contain adventitious agents, cell cultures represent
uncertain hazards and should be treated as potentially
infectious (ie as category 2 agents) even when an infectious
agent has not been identified (ie the cells have not been
tested for contamination). Class II containment requires the
use of an appropriate safety cabinet, spill resistant
benching, documented disinfection procedures and a restricted
access to the laboratory area. Containment of organisms being
transported between laboratories is extremely important since
it introduces the possibility of direct transmission of
organisms to the general public. Regulations relating to the
transportation of biological agents within Europe have been
recently updated (eg CHIPS II 1994 for UK (3)) and for global
transportation IATA (8) have also prepared new regulations.
Laboratories distributing biological material also have
responsibilities to the recipient of cells or other
potentially infectious material. The person receiving the
culture should be supplied with adequate information; firstly,
to enable them to recognise that the material is correct (ie a
detailed physical description of the package contents) and
secondly, to assist them in the preparation of their own risk
assessments. This information can be provided as a materials
safety data sheet (a statutory requirement in the UK) which
not only identifies hazard but also describes the physical
appearance of the material supplied. Thus, loss of material
in transit or provision of the wrong material can be
identified immediately. In addition the distributor of the
culture must ensure that all recipients have appropriate
facilities and trained staff for its safe handling.
CONCLUSION
In general, establishing relative theoretical risks of animal
cell cultures in use is straightforward. This will be
adequately addressed in most cases by the use of the European
containment level 2 precautions and the use of a safety
cabinet which protects the operator. Infection of a cell line
with hazardous organisms automatically requires that
containment should be appropriate for the organism in
question. Additional assessment of manipulation and disposal
procedures is important to identify and contain any aspect
which exposes the operator to unreasonable risks. The
application of good safety procedures can be used to give
benefits within the laboratory far beyond the obvious
requirement for healthy staff. Careful risk assessment
respecting scale of work and the whole procedure (in addition
to individual assessment of agents and reagents) will ensure
safe working conditions for laboratory staff and if applied
properly also encourages clean, efficient and well documented
work procedures which are synonymous with good science and
economical use of time and resources.
REFERENCES
1 ACDP- Categorisation of biological agents according to
hazard and categories of containment. Fourth edition, 1995,
Advisory Committee on Dangerous Pathogens, HSE books, PO Box
1999, Sudbury, Suffolk, C010 6FS, UK.
2 Anon. British Standard BS 5726 (four Parts), 1992. British
Standards Institution, 386 Chiswick High Road, London, UK.
3 CHIPS II. Chemicals (Hazards Information and Packaging for
Supply) Regulations 1994. HSE books, Sudbury, UK.
4 Del Giudice R.A and Gardella R.S, 1984. Mycoplasma
infection of cell cultures: Effects, incidence and detection.
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Gaithersburg, USA.
5 Erickson G.A., Bolin S.R. and Landgraf J.G., 1991. Viral
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6 Frommer W., Archer L., Boon B., Brunius, G., Collins, C.H.,
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7 Hay R.J, 1991. Operator-induced contamination in cell
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8 IATA Dangerous Goods Regulations, 1995. Thirty fifth
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9 Mahy B.W.J., Dykewicz, C, Fisher-Hoch, S, Ostroff, S.,
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11 Stacey, G.N. and Sheeley, H.J., 1994. Have biosafety
issues in cell culture been overlooked. J. Chem. Tech.
Biotechnol. 61: 95-96.
Published by Bioline Publications Ltd and Science and
Technology Letters.
Copyright held by the author.
Editorial Office: biosafe@biostrat.demon.co.uk
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