Tissue culture waste disposal guide Dr. Pornsawan Leaungwutiwong (Tropmed MU) Lab Safety Training 16-17 January 2017

1. Animal cell cultures : Risk assessment and
biosafety recommendations
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2. Animal cell cultures
Risk assessment and biosafety recommendations
• The use of animal and human cell cultures has become very beneficial for diverse
applications in biotechnology and biomedical research.
• Originally used as substrates for the production of viral vaccines, animal and human cell
cultures became an indispensable tool to produce a variety of products, including
biopharmaceuticals, monoclonal antibodies and products for gene therapy. The use of animal
and human cell cultures constitute also adequate test systems for studying biochemical
pathways, virus production, pathological mechanisms or intra- and intercellular responses.
• Along with the increasing importance of the contained use of animal and human cell
cultures, biosafety concerns have pointed to the risks with respect to human health and
environmental considerations.
• A maximal reduction of these risks necessitates a thorough biosafety risk assessment, taking
account of the type of manipulation and the biological hazards inherent to the use of cell
cultures.
• The risk assessment should result in the implementation of appropriate containment
measures and work practices in order to provide maximal protection of human health and
environment
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3. Risk assessment of the infecting pathogen
Risk assessment of the infecting pathogen
should consider :
• Pathogen characteristics (description of the
microorganism)
• Host range
• Pathogenicity
• Mode of transmission
• Infectious dose (if known)
• Communicability
• Epidemiology (the assignment of appropriate risk
group may depend on the geographic localization)
• Reservoir
• Vectors
• Zoonosis
• Drug susceptibility
• Susceptibility to disinfectants
• Survival outside host
• Physical inactivation
Along with aspects related to the disease
caused by the pathogen , such as :
• Treatment
• Immunization
• Prophylaxis
• Laboratory-acquired infection
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4. The level of risk of animal cell culture
Low risk - Non human/non primate continuous cell lines and some
well characterized human continuous lines.
Medium risk - Poorly characterized mammalian cell lines.
High risk - Primary cells derived from human/primate tissue or blood
- Cell lines with endogenous pathogens (the precise
categorization is dependent upon the pathogen)
- Cell lines used following experimental infection where the
categorization is dependent upon the infecting agent – refer
to ACDP guidelines, for details.
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5. Working with cell line
• A culture collection such as ECACC will recommend a minimum
containment level required for a given cell line based upon its risk
assessment. For most cell lines the appropriate level of containment
is Level 2 requiring a class 2 microbiological safety cabinet.
• However, this may need to be increased to containment Level 3
depending upon the type of manipulations to be carried out and
whether large culture volumes are envisaged.
• For cell lines derived from patients with HIV or Human T-
Lymphotropic Virus (HTLV) Level 3 containment is required.
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6. Working with cell line
• Containment is the most obvious means of reducing risk.
• Other less obvious measures include restricting the movement of staff and
equipment into and out of laboratories.
• Good laboratory practice and good bench techniques such as ensuring
work areas are uncluttered, reagents are correctly labelled and stored, are
also important for reducing risk and making the laboratory a safe
environment in which to work.
• The risk of exposure to aerosols or splashes can be limited by avoiding
rapid pipetting, scraping and pouring.
• In addition, it is recommended that people working in laboratories where
primary human material is used are vaccinated against Hepatitis B. Staff
training and the use of written standard operating procedures and risk
assessments will also reduce the potential for harm.
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7. Deliberate infection with pathogens
• In this case, the determination of potential hazards related to infected
cell cultures requires an examination of the inherent properties of the
infecting pathogen.
• This implies an assessment of a list of criteria, which are specific to
the pathogen, along with aspects such as the existence of effective
therapies or prophylaxis.
• An evaluation of these criteria have been used to classify pathogens
into classes of biological risk, also called risk groups.
• The biological risk of infected cell cultures will depend on the
biological risk of the infecting pathogen(s).
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8. Adventitious contaminating agents
• When manipulating cell cultures, the presence of adventitious
contaminating agents constitutes the main hazard to humans.
• Contamination may occur by the source (e.g. infected animals or
tissues), during cell handling (repeated passages and use in the
laboratory)
• Using contaminated biological reagents (e.g. media additives
derived from bovine sources are often contaminated with bovine
viral diarrhoea virus, BVDV).
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9. Adventitious contaminating agents
Bacteria and Fungi
• In general bacterial or fungal contamination can be
readily detected in cell cultures because of their
capacity to overgrow cell cultures. Bacterial and
fungal infections are relatively easy to prevent and
to treat
Parasites
• Adventitious contamination with parasites may be
an issue when handling freshly prepared primary
cell cultures or tissue cultures originating from a
donor organism that is known or suspected to be
infected with a specific parasite.
• Well-known intracellular protozoan parasites for
which laboratory-acquired infections have been
reported are Toxoplasma gondii, Trypanosoma
cruzi, Leishmania sp, Cryptosporidium
parvum , Plasmodium sp. etc.
(reviewed by Herwaldt et al., 2001).
Mycoplasma
• Compared to bacterial or fungal infections,
contaminations with mycoplasma give more
problems in terms of incidence, detectability,
prevention and eradication.
• Mycoplasma infection may go undetected for many
passages, causing a variety of unpredictable effects
causing harm to the host cell.
• Mycoplasma infection may also influence the
sensitivity of host cells for growth of viruses.
• Beside the fact that this intracellular bacteria is
one of the most common cell culture
contaminants, it should be considered that some
of contaminating Mycoplasma spp. belong to class
of risk 2.
• Together with M. arginini, M.orale,
M.pirum and M. fermentans, pathogenic organisms
like M. gallisepticum (class of risk 3 for animals), M.
hyorhinis (class of risk 2 for animals), and M.
pneumoniae (class of risk 2 for humans) account
for more than 96% of mycoplasma contaminants in
cell cultures.
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10. Adventitious contaminating agents
Viruses
• Viral contamination needs particular
attention because infection may be without
cytopathic effect for the cell culture or may
be latent (e.g. herpesvirus) and hard to
detect.
• Human and non-human primate cultures are
more likely to harbor viruses that are highly
pathogenic to humans.
• Of particular concern are the blood-borne
viruses such as Hepatitis B Virus (HBV),
Human Immunodeficiency Virus (HIV) and
others such as Hepatitis C virus (HCV) and
Human T-cell lymphotropic viruses (HTLV).
• However, non-human cell cultures are not
without risks as they may contain viruses with
a broader host range able to infect humans
such as rodent cell culture carrying hantavirus
(Lloyd G & Jones N, 1986) or primate cells
harbouring Marburg virus.
Lymphocytic choriomeningitis virus (LCMV)
contamination of murine cell cultures
• Lymphocytic choriomeningitis virus (LCMV) is an
arenavirus that establishes a silent, chronical infection
in mice but causes aseptic meningitis, encephalitis or
meningoencephalitis to humans.
• LCMV is a potential contaminant of cell cultures. The
significance of LCMV contamination has been
reinforced by the description of cases of laboratory-
acquired LCMV infections arising from contaminated
murine tumor cell lines (Mahy, et al. 1991).
• Laboratory workers that manipulate infected rodents
or cell cultures originating from infected animals
expose themselves at risk by directly exposing cuts,
open wounds or mucus membranes with infected
body fluids .
• The risk can be minimized by utilizing animals or cell
cultures from sources that are regularly tested for the
virus.
• Manipulation of LCMV infected material or material
with an increased likelihood of LCMV contamination
necessitates the implementation of a biosafety
containment level 2 (L2).
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11. Animal cell cultures : Precautionary measures
• Respect good microbiological practices, especially those that are aimed at avoiding accidental
contamination
• Treat each new culture that is manipulated for the first time in the laboratory facility as
potentially infectious.
• The use of a biosafety cabinet of class type II is strongly recommended until the cells have been
shown negative in sterility tests for bacteria (including mycoplasma) and fungi.
• Cell cultures from ill-defined sources should be handled under biosafety level 2 (L2) conditions. ]
• If there is a reasonable likelihood of adventitious agents of higher risk class, the cell line should
be handled under the appropriate containment level until tests have proven safety.
• Clean up any culture fluid spills immediately
• Work with one cell line at a time, disinfect the work surfaces between cell lines handling, aliquot
growth medium. So that the same vessel is not used for more than one cell line.
• Avoid pouring actions that are a potential source of cross-contamination.
• If necessary, carry out a quality control of cells demonstrating the absence of likely
contaminating pathogens (e.g. PCR, reverse transcriptase detection of suspected pathogen).
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12. Tissue Culture Waste Disposal
Guide
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13. Tissue Culture Waste Disposal Guide
• The culturing and preparation of cells or tissues generates liquid
wastes that must be assessed and managed according to the
biological and chemical hazards contained within them.
• Potentially infectious cell or tissue culture waste media constitutes a
biological waste that must be disinfected prior to disposal.
• The Centers for Disease Control & Prevention (CDC) recommend that
the vacuum systems used to aspirate these wastes are protected with
High Efficiency Particulate Air (HEPA) filters and liquid disinfectant
traps to prevent aerosolized microorganisms from being emitted into
the laboratory or the system exhaust.
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14. 1. Utilizing thick-walled plastic tubing, aspirate the
media from the sample flask to the aspiration
vacuum flask
(A).The aspiration vacuum flask (A) is connected to
the overflow flask (B) an in-line HEPA filter (C) is
used to protect the vacuum system (D).
The left aspiration flask (A) is used to collect the
contaminated fluids (tissue culture media, stains,
fixative) into a suitable decontamination solution;
the right flask (B) serves as a fluid overflow
collection vessel.
Please note that the addition of a glass or plastic
tube into the solution in flask B minimizes aerosols.
 Filters must be replaced as needed—at a
minimum, annually and when there is any evidence
of deficiencies (e.g. filter blockage, failure,
wetness).
When changing out filters, dispose of them as
regulated medical waste, hazardous chemical
waste, or radiological waste, as appropriate.
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15. 1. ADD DISINFECTANT: Fill the Aspiration Vacuum Flask
(A) with bleach to ~10% of the flask’s volume.
2. If a different EPA-approved disinfectant is utilized,
add the volume of disinfectant required to achieve the
manufacturer’s recommended concentration.
3. When bleach and water are mixed together, the
solution’s disinfectant qualities only last 24 hours.
Additional bleach may be required.
4. LABEL: Label both flasks with a Tissue Culture Waste
Label, and indicate the constituents of the mixture;
including the type of tissue culture media, disinfectant
used, and other chemical constituents.
5. CONTAINMENT: Place the vacuum flasks in secondary
containment (e.g., bin or tray) if outside a biosafety
cabinet to hold the liquid if it is spilled or released.
6. ASPIRATE: Aspirate the tissue culture waste into the
flask containing disinfectant (A). Discontinue use when
the vacuum flask is 75% full.
7. ADD ADDITIONAL DISINFECTANT: Add an additional
volume of disinfectant required to achieve the
manufacturer’s recommended concentration (e.g., 10%
bleach).
8. STIR: Stir to mix, then leave at room temperature for
2 hours, or let sit overnight to ensure sufficient contact
time with disinfectant.
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16. Waste Disposal
• Tissue culture waste (culture medium) – inactivate for at least 2 hours
in a solution of hypochlorite (10,000ppm) prior to disposal to drain
with an excess of water.
• Contaminated pipettes should be placed in hypochlorite solution
(2500ppm) overnight before disposal by autoclaving and incineration.
• Solid waste such as flasks, centrifuge tubes, contaminated gloves,
tissues, etc., should be placed inside heavy-duty sacks for
contaminated waste and incinerated.
• If at all possible waste should be incinerated rather than autoclaved.
• Waste from specially licensed laboratories e.g. those handling
genetically modified level 3 (GM3) organisms requires specific
treatment and tracking.
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17. References
• Safety Aspects of Cell Culture, Sigma-Aldrich
http://www.sigmaaldrich.com/technical-
documents/protocols/biology/safety-aspects-of.html
• Tissue Culture Waste Disposal Guide, Weill Cornell Medicine
http://weill.cornell.edu/ehs/static_local/pdfs/Tissue_Culture_Waste.pdf
• Belgian biosafety server, Animal cell cultures : Risk assessment and
biosafety recommendations
http://www.biosafety.be/CU/animalcellcultures/mainpage.html
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