Department of Physiology
Procedure No:
PHY/SOP/RE02
Revision No:
001
Title:
Effective Date :
14/6/2010
Safe Handling of Mammalian Cell Cultures
(Adopted from OSHE/SOP/BS/08)
Page:
Page 1 of 7
Safety & Health
Laboratory (Location):
Cytokine biology Lab, MD9
Prepared/Review By
Approved By
Next Review Date
Oh Chu Yun
Dr. Leung Pui Lam Bernard
14/6/2013
* Review Date = Future date for the next revision (every 3yrs)
1.0 OBJECTIVE
The objective of this SOP is to provide guidance to all laboratory personnel on the safe handling
of mammalian cell culture to prevent accidental exposures to infectious agents / toxic or harmful
materials.
2.0 SCOPE
The procedure is applicable to all Department of Physiology laboratories which carry out the
procedures of mammalian cell cultures. All staff and students are to adopt the practices in the
procedure.
3.0 RESPONSIBILITIES
3.1 Head of Department/ Principal Investigator
Principal Investigators (PI) and Heads of Department (HOD) shall be responsible for
ensuring that this SOP is disseminated to all laboratory personnel and that they are
aware of the procedures to take for safe handling of mammalian cell cultures.
3.2 Laboratory personnel
All lab personnel shall be responsible for proper handling and use of mammalian cell
cultures in accordance with this procedure. All lab personnel shall report any spills or
mishaps to their supervisor or the Faculty Safety and Health officer.
4.0 DEFINITION
Nil
Department of Physiology
Procedure No:
PHY/SOP/RE02
Revision No:
001
Title:
Effective Date :
14/6/2010
Safe Handling of Mammalian Cell Cultures
(Adopted from OSHE/SOP/BS/08)
Page:
Page 2 of 7
Safety & Health
Laboratory (Location):
Cytokine biology Lab, MD9
5.0 PROCEDURE
Manipulations of mammalian cell cultures pose a risk to lab personnel. Major hazards
associated with the manipulation of animal cell cultures:
a. Risk of exposure of the worker to biological agents,
b. Risk involved with the use of genetically-modified cell lines,
c. Risk from adventitious contamination of cell cultures. (Animal cell cultures may provide a
support for contaminating adventitious bacteria, fungi, mycoplasma or viruses that cause
harm to human health)
5.1 Risk Assessment
a. Evaluate the risks associated with each cell line to be handled. Prepare safety
protocols for the appropriate use, storage, decontamination, disposal, and
emergency response procedures for the cell line used.
b.
c.
The risk assessment should involve an evaluation of:
the intrinsic properties of the cell culture,
subsequent properties acquired as a result of any genetic modification,
the possibility that the cell culture may inadvertently or deliberately become
contaminated with pathogens, and
the type of manipulation
More information on risk assessment and precautions can be found in the
references (1-2). The article: “Animal Cell Cultures: Risk Assessment and
Biosafety Recommendations” (1) gives a detailed consideration of each of the
factors described above while the CDC publication-“Biosafety in Microbiological
and Biomedical Laboratories (BMBL)” (2) gives information on agent-specific risk
assessment.
5.2 Containment level
a. As a rule, for cell cultures known to harbor an infectious etiologic agent, manipulate
the cultures in compliance with containment measures recommended for the
etiologic agent.
b.
No cell line is guaranteed to be non-hazardous. When handling human cell lines,
blood or tissue, assume that infectious agents are present. Work with cell cultures
from human or primate origin should minimally be performed under Biosafety Level
2 practices and containment.
Department of Physiology
Procedure No:
PHY/SOP/RE02
Revision No:
001
Title:
Effective Date :
14/6/2010
Safe Handling of Mammalian Cell Cultures
(Adopted from OSHE/SOP/BS/08)
Page:
Page 3 of 7
Safety & Health
Laboratory (Location):
c.
-
Cytokine biology Lab, MD9
In every case, containment measures should minimize adventitious contamination
of the cell cultures and offer a maximal protection of human health and the
environment. A class II Biological Safety Cabinet (BSC) can be found in almost
every tissue culture lab. It is designed to:
Protect you from being exposed to infectious aerosols that may be generated
within the cabinet.
Protect cultures from microbial contaminants that are ubiquitous in room air.
d. When working with a recombinant cell line, risk assessments must consider the
hazards associated with the vector used for the transformation and the insert. If the
vector or insert introduces additional risk, a relatively safe cell line may require a
higher containment level.
e. (Refer to Appendix 1 for guidance on assignment of containment level.)
5.3 Safe Practices
a. Practice good microbiological practices, especially those that are aimed at
avoiding accidental contamination. Be trained in aseptic techniques
b. Wear a long –sleeved lab coat, gloves and personal eye protection. Maintain a
clean lab coat reserved solely for cell culture work.
c. Remove contaminated gloves carefully. Wash hands thoroughly with antiseptic
soap after removing gloves.
d. Keep the work area clean and tidy
e. Avoid the use of sharps.
f. Avoid the creation of aerosols through vigorous pipetting.
g. Never lay the pipette on its side when it is filled with liquid.
h. Sterile handling in a biosafety cabinet (BSC):
Swab down work surfaces and materials with 70% ethanol before and after
use.
Plan ahead and take all needed supplies to the BSC before beginning work.
Delineate areas for clean and dirty materials.
Avoid talking during culture manipulations as aerosols may be drawn into the
work area.
Avoid pouring actions, which are a potential source of cross-contamination.
Do not place contaminated tubes, pipettes on work surface.
Do not leave media bottles or culture vessels open.
Discard unwanted, non-sterile tubes or pipettes immediately. Place a
horizontal pan/ tub filled with an appropriate disinfectant to disinfect
contaminated pipettes before discard.
Department of Physiology
Procedure No:
PHY/SOP/RE02
Revision No:
001
Title:
Effective Date :
14/6/2010
Safe Handling of Mammalian Cell Cultures
(Adopted from OSHE/SOP/BS/08)
Page:
Page 4 of 7
Safety & Health
Laboratory (Location):
Cytokine biology Lab, MD9
i.
Cell Lines:
Use well characterized cell lines where possible.
Read all information such as Material Safety Data Sheets provided with the
cells and ensure you are aware of the hazards and precautions for handling.
- Treat each new culture that is manipulated for the first time in the laboratory
facility as potentially infectious.
- Handle cell cultures from undefined sources as risk group 2 agents. If there
is a reasonable likelihood of adventitious agents of higher risk class, the cell
line should be handled under appropriate containment level until tests have
proven safety.
- NEVER handle or manipulate cells derived from yourself. Autologous cells,
if accidentally reintroduced, can evade normal immune responses.
- Quarantine new cell cultures brought to the laboratory until the culture does
not show growth of contaminating bacteria, fungi, or mycoplasmas.
- Work with one cell line at a time and disinfect the work surfaces between
two handlings involving cell lines.
- Routinely carry out quality control checks of cells to ensure the absence of
likely contaminating pathogens.
j.
Culture media:
When preparing media to be used with more than one cell line, aliquot the
media into separate containers for each cell line. This will reduce the
chances of contamination.
Restrict use of antibiotics in growth media to reduce the chance of a
resistant agent from growing. Rely on good aseptic technique rather than
on antibiotics to keep cell lines free of contamination.
Obtain animal sera from a reputable source as serum can be contaminated
with adventitious bovine viruses or prions eg. the agent responsible for
bovine spongiform encephalopathy (BSE) or “mad cow disease”.
k. Use of equipment:
Perform routine verification of autoclave working condition. Refer to
CBL/SOP/05- “Operation of Autoclaves” for details.
Do not use laminar flow benches when using cell lines containing infectious
agents. Refer to SOP (CBL/SOP/09) – “Safe use of laboratory equipment”
for more information on the proper use of various laboratory hoods.
Ensure you understand the correct operation of a biological safety cabinet.
Refer to SOP (CBL/SOP/08) - “Safe operation of Biosafety Cabinets”.
Protect all vacuum lines used for aspirations of cultures, with disposable air
filters and/or liquid traps. Refer to SOP (CBL/SOP/09) – “Safe use of
laboratory equipment” for more information.
Department of Physiology
Procedure No:
PHY/SOP/RE02
Revision No:
001
Title:
Effective Date :
14/6/2010
Safe Handling of Mammalian Cell Cultures
(Adopted from OSHE/SOP/BS/08)
Page:
Page 5 of 7
Safety & Health
Laboratory (Location):
l.
Cytokine biology Lab, MD9
Decontamination:
Decontaminate all cell culture wastes by autoclaving or with appropriate
disinfectant according to SOP (CBL/SOP/04) “Biological Waste Disposal”
Decontaminate all reusable glass and plastic ware immediately after use.
Do not allow a lab worker/dishwasher to be exposed to contaminated lab
ware.
Clean up any culture fluid spills immediately with a validated disinfectant.
5.4 Working with Human and Other Primate Cells
a. The potential laboratory hazards associated with human cells include:
- blood borne pathogens e.g. Hepatitis B virus and HIV,
- cells transformed with oncogenic viruses agents, such as SV-40, EBV, or HBV, as
well as cells carrying viral genomic material,
- tumorigenic human cells with potential hazards as a result of self-inoculation
- Contamination with adventitious bacteria, fungi, mycoplasma, and viruses that
cause disease in humans.
b. Handle all human and primate cells using Biosafety Level 2 practices and containment.
Since most cell lines are not fully characterized, it is wise to regard such cell lines as
potentially infectious and handle them at the same biosafety level as a cell line known to
carry HIV or hepatitis B virus.
c. Perform all work in a biosafety cabinet when handling infectious materials, or when there
is a potential of aerosol production. Decontaminate all material by autoclaving or
disinfection before discarding.
d. A Hepatitis B immunization is required for all personnel handling human cell lines.
e. All established or permanent cultures of human lymphocytes should be handled on the
assumption that they harbor the Epstein-Barr virus.
6 RECORDS
a. Records for the disposal of the wastes must be kept for verification by authorities or for
inspection.
b. Records of equipment certification / maintenance schedules should be kept on file.
Department of Physiology
Procedure No:
PHY/SOP/RE02
Revision No:
001
Title:
Effective Date :
14/6/2010
Safe Handling of Mammalian Cell Cultures
(Adopted from OSHE/SOP/BS/08)
Page:
Page 6 of 7
Safety & Health
Laboratory (Location):
Cytokine biology Lab, MD9
7 REFERENCES
1. Pauwels, K et al. (2007) Animal Cell Cultures: Risk Assessment and Biosafety
Recommendations. Applied Biosafety, 12(1) pp. 26-38
2. National Institutes of Health, Department of Health and Human Services, USA, 2007.
Biosafety in Microbiological and Biomedical Laboratories (BMBL), 5th edition
(http://www.cdc.gov/OD/ohs/biosfty/bmbl5/bmbl5toc.htm)
3. CBL/SOP/05 - “Operation of Autoclaves
4. CBL/SOP/08 -“Safe Operation of Biological Safety Cabinets”
5. CBL/SOP/09 - “Safe Use of Laboratory equipment”
Department of Physiology
Procedure No:
PHY/SOP/RE02
Revision No:
001
Title:
Effective Date :
14/6/2010
Safe Handling of Mammalian Cell Cultures
(Adopted from OSHE/SOP/BS/08)
Page:
Page 7 of 7
Safety & Health
Laboratory (Location):
Cytokine biology Lab, MD9
8 APPENDICES
Appendix 1 Guidelines for selection of containment level for cell lines.
(Adapted from reference 2)