Cell Culture Safe Work Standard
Intent and Application ... 3
Risk Group and Containment Level ... 3
Safety Requirements ... 4
Engineering Controls (required equipment) ... 4
Administrative Controls (training) ... 4
Administrative Controls (Standard Operating Procedures) ... 4
Personal Protective Equipment (minimum required) ... 4
Vaccination ... 4
Hazards and Safety Practices ... 5
Unidentified Pathogenic Contaminants ... 5
Extracorporeal Metastasis (3) ... 7
Liquid Nitrogen ... 8
Intent and Application
The intent of this standard is to state the safety requirements for workers conducting cell culture work in order to mitigate hazardous exposure. It highlights hazards that may be encountered and provides safety practices to reduce exposure risks.
This standard applies to the use and/or propagation of clinical cell isolates, primary cell cultures, hybridoma cell lines, and continuous or immortalized cell lines hereafter collectively referred to as “cells”.
Risk Group and Containment Level
The Manitoba Workplace Safety and Health Act and Regulation are the legal statutes that apply to the use of cell lines in the workplacei (1).
At the University of Manitoba work with cells is classified Risk Group 2 Unregulated which means that work with cells poses a moderate individual risk and low community risk. Work with cells will be carried out according to Containment Level 2 Physical Containment and Operational Practice Requirements for biosafety as described in the Canadian Biosafety Standard and the University of Manitoba Biosafety Manual. Depending on tissue source and competency, the cell lines are further classified by risk (Appendix 1).
Some suppliers or manufacturers may identify their cells as Risk Group/Biosafety Level 1; however, all cells used in Biosafety Permitted facilities at the University of Manitoba Biosafety will be manipulated as Risk Group 2 agents. Use the Cell Culture Risk Assessment Worksheet (Appendix 2) if your cell line does not have a material safety data sheet.
Special Note:
• The use of blood or tissue from lab staff for the development of transformed
cell lines is not permitted due to the increased risk to lab workers
subsequently exposed to these cell lines. The subsequently exposed worker
may have severely reduced resistance to tumorigenesis caused by their own
transformed cells (2).
Safety Requirements
Engineering Controls (required equipment)
Biological safety cabinet for all aerosol generating procedures (sonication, homogenization, pipetting etc)
Sealed cups or rotors on centrifuges
Safety engineered sharps
Administrative Controls (training)
University of Manitoba Generic Biosafety Training
Site Specific Biosafety Training (lab specific)
Aseptic technique training (lab specific)
Administrative Controls (Standard Operating Procedures)
Biological Safety Cabinet decontamination procedure (lab specific)
Incubator decontamination procedure (lab specific)
Personal Protective Equipment (minimum required)
Pants that cover legs
Shoes with closed toe and heel
Lab coat with knitted cuffs and snap closures (white coats with open sleeves and buttons are not acceptable)
Disposable gloves
Safety eyewear
Thermal insulated gloves and face shield for work with liquid nitrogen
Vaccination
Hepatitis B required for workers working with human clinical specimens and isolates, recommended for work with established human cell lines. Not required for work with animal primary or established cell lines.
Hazards and Safety Practices
Unidentified Pathogenic Contaminants
Unidentified and/or undocumented pathogens carried by the cells or components of the cell culture medium are the main biological hazard posed by cell culture work. Cells may carry undocumented viral pathogens and sera could contain microorganisms including viruses and mycoplasma.
Cell lines may be screened for the human blood borne pathogens most commonly considered to be a risk to workers, namely Hepatitis and Human Immunodeficiency Virus. Lab supervisors and workers should also take into account other pathogens which may be present in human origin cell samples such as Influenza, West Nile Virus, malaria, sexually transmitted infections, Lyme Disease, and any other acute or chronic infection which may be present in humans. Screening of cell lines does not commonly include all possible pathogenic contaminants; the process would not be practical.
Quality Assurance
Assumptions must not be made about pathogen screening for cells purchased from commercial suppliers. Quality assurance/testing documentation specific for the lot of cells purchased should be provided by the supplier. If documentation is not available or it doesn’t specifically state what pathogen testing has been carried out then pathogen testing has not been completed. Similarly, when acquiring cells; from research collaborators, if quality assurance/testing documentation has not been provided, then testing has not been carried out. If cells have been grown or manipulated in the collaborating lab the original quality assurance documentation is no longer valid. Cells should therefore be handled with precautions as though pathogenic contaminants are present.
The health status of a cell donor/source cannot be used as proof that pathogens are not present in clinical samples. Contamination can occur at various points along the harvest and culture process and blood tests/clinical symptoms may not reveal the microbiological status of donated tissues.
Mycoplasma Contamination (2)
Mycoplasma contamination is frequent in cell culture, they are unaffected by many antibiotics used in cell culture, and due to a lack of visible signs, the contamination can go unnoticed. Mycoplasmas can cause serious respiratory illness as well as a variety of other pathogenic effects such as inflammatory arthritis, joint infection, central nervous system infection, among others. Hazardous routes of exposure include inhalation of infectious aerosols and contact with mucosal membranes. Mycoplasma infection is communicable between humans (4,5). Refer to the attached pathogen safety data sheets for more information.
Mycoplasma contamination can occur through many routes during routine cell culture work including cross contamination from positive cultures, contaminated equipment or
work surfaces, culture media such as sera and reagents, lab animals, and infected lab staff.
Preventative measures rely mainly on aseptic cell culture technique which requires significant training and experience of lab workers. Frequent decontamination of biological safety cabinets, incubators, use of inline filters on carbon dioxide supply lines, and the use of sterile water to humidify incubators will help reduce the presence of mycoplasmas on work surfaces. Effective disinfectants include 70% ethanol and 1% sodium hypochlorite (4). Other helpful measures include isolation of cell cultures and media, quarantine procedures for new cell lines in the lab, and routine testing for mycoplasma contamination.
Bacterial and Fungal Contamination (2)
Aseptic technique is a must to prevent bacterial/fungal contamination. The routine use of antibiotics to control bacterial growth should be avoided in favor of discarding contaminated cultures. This prevents the creation of antibiotic-resistant bacterium and avoids propagating co-cultures of resistant bacteria that may be present at low levels. Bacterial or fungal contamination can be detected through:
direct observation of increased turbidity (cloudiness in the medium)
change in pH (change in colorimetric pH indicators in the culture media).
microbial culture or microscopic observation using Gram’s stain.
If cells are irreplaceable, it would be necessary to conduct resistance screening on the contaminating agent to find a useable antibiotic, treat the culture, and establish an uninfected culture. Wide spectrum antibiotic resistance may make this process especially difficult.
Viral Contamination
Pathogenic viruses may be present in cell culture samples. The viral contaminants may originate with the source of the cells or may make their way in as part of the cell culture process. Serious lab acquired infections such as hantavirus and lymphocytic choriomeningitis virus, have occurred due to exposure to tissues and cells cultures taken from rodents. For this reason all animals used must be specific pathogen free (2).
Sources which may introduce viral contaminants can include any reagents used in the culture process that are derived from animals such as serum. Cells may also become contaminated when they are implanted into animals and harvested afterward. Since there are no universal testing systems it is difficult to verify whether cell lines are contaminated.
Extracorporeal Metastasis (3)
Extracorporeal metastasis is described as human-to-human cancer transmission through cancer cells. Transmission of metastatic cancer has been demonstrated experimentally in humans, during surgical incidents, and in lab-acquired infections. The precedents above were caused by direct transplantation or puncture injuries; however, it is reasonable to assume that human-to-human cancer transmission could occur through cracks in the skin, mucosal membranes, and by inhalation.
It is felt that human-to-human cancer transmission is either unknown or dismissed as implausible in normal lab practice. Growth of tumor cells from another individual is unlikely in a healthy worker but those with compromised immunity may be at increased risk.
•Aerosols, splashes to the skin and eyes, puncture injuries Routes of Exposure:
•Orient all workers working with cell lines to be cognizant of the risks posed by
undocumented pathogens present in cell cultures of all types. Discourage complacency brought about by assumptions about the presence of pathogens in original samples, and the expectation that consistent supplementation with antibiotics will take care of all pathogen risks.
•Purchase reagents and sera from suppliers that issue certificates of analysis/quality assurance documentation with each batch of product. Retain copies of quality assurance documentation as part of lab documentation (electronic copies are suitable).
•Train workers on aseptic technique to reduce sources of contamination and ensure effective decontamination procedures are in place for biological safety cabinets and incubators. Safety Practices:
•Puncture injuries, splashes to the skin and eyes Routes of Exposure:
•The most important safety measure which must be put in place is to recognize that there is a potential direct hazard posed by cancer cell lines/immortalized/transformed cell themselves and to train lab workers to recognize that hazard. Advise workers that all cell lines should be treated as biologically hazardous and that there is a potential for lab acquired metastasis while working with these materials. The intent of this advice is not to prohibit work but rather to appropriately warn workers of foreseeable dangers. If directly exposed, workers must treat these exposures as hazardous and follow the University Post Exposure Protocol or equivalent.
•Use safety engineered sharps to reduce or eliminate the use of tools that can puncture the skin such as syringes, scissors, needles, razors, and scalpels.
Liquid Nitrogen
The most common, acute, daily hazard found in cell culture is the near universal use of liquid nitrogen for storage of cell lines. Liquid nitrogen is hazardous due to the extremely low temperature and can cause frostbite and tissue damage virtually instantly upon direct contact with the skin and/or eyes. It can also cause explosions if contained in an unvented/closed container due to expansion of the liquid to a gaseous state as it is warmed. Nitrogen gas also poses a risk as an asphyxiate (can suffocate a person) if it is used in sufficient volumes in an area such as a cold room or other area without sufficient fresh air supply. In addition to the safety equipment listed above workers must wear thermal insulated protective gloves and a face shield while working with liquid nitrogen or retrieving samples from liquid nitrogen storage.
References
1. Manitoba Workplace Safety and Health Act C.C.S.M c. W210, Workplace Safety and Health Regulation 217/2006 M.R. 128-2019
2. Guidelines for the use of cell lines in biomedical research. R. J Geraghty et. al. British Journal of Cancer (2014) 111, 1021–1046
3. Comment on: ‘Guidelines for the use of cell lines in biomedical research’: human-to-human cancer transmission as a laboratory safety concern. Y. Lazebnik and G. E. Parris - British Journal of Cancer (2015) 112, 1976–1977
4. Pathogen Safety Data Sheets: Infectious Substances – Mycoplasma spp. Government of Canada Date modified: 2011-02-18
5. Pathogen Safety Data Sheets: Infectious Substances – Mycoplasma hominis. Government of Canada Date modified: 2011-02-18
• Inhalation, splashes to the skin and eyes, and traumatic injury. Routes of Exposure:
• Do not store sample tubes in the liquid phase of nitrogen. Wear safety eye-wear when thawing any ultra-cold temperature stored materials, even when working in a biological safety cabinet. An incident occurred at the University of Manitoba where a worker was thawing a container held at -80oC in the biological safety cabinet. The tube failed catastrophically with sufficient energy that a piece of the broken tube ricocheted off the work surface and struck the worker directly in the eye.
Human, Bovine,
Ovine Central
Nervous System
Tissue (Prion
Concern)
Human primary
cells/tissues/blood
and body fluids
Cells/Tissues from SPF-free
lab-bred animals
Established Human Cell lines
Established animal cell lines
Incr
easin
g
Risk
Biosafety Officer Steven Cole P310 Pathology Bldg Winnipeg, Manitoba Canada R3E 0W3 Tel: (204) 789-3675 Biosafety Specialist Vanessa Pinto P310 Pathology Bldg Winnipeg, Manitoba Canada R3E 0W3 Tel: (204) 789-3477
Biological Safety Program
Cell Culture Risk Assessment Worksheet
A. Pathogen Risk Assessment
• Cell Culture/Line Name: • Cell Type:
Primary
Established/Continuous Hybridoma Cell line Other
• Species of Origin: Human
Non-human primate Other:
• Organ/ Tissue of origin:
• Identify if obtained From Commercial source: (e.g. ATCC) o Catalogue Number:
• Identify if obtained from another source :
• Transformed? Yes No
o If yes – specify: (e.g. chemical, oncogene, human oncogenic virus)
• Has a quality assurance verification for the the cell line been performed? (Has the cell culture been tested for any potential pathogens?) Yes No Don’t Know
o If yes, how and what was assessed, when, and by whom?
• Is the cell culture is known to be infected with a pathogen? Yes No Don’t Know
o If yes – Name:
o Risk Group 1 2 3
• Is a vaccine available for the identified pathogen? Yes No • Is an MSDS available for the pathogen(s)? Yes No Not Applicable
o If yes, then also review the specific pathogen risk assessment checklist.
• Are toxins or viral particles actively secreted? (E.g EBV) Yes No o If yes name
B. Personnel Risk Assessment
• Have all lab personnel working with or near any of the above material received
documented training as per this risk assessment as well as a program-specific training, describing the routes of exposure, signs/symptoms of disease, treatments, U of M Post Exposure Protocol and the requirement to report for medical attention if symptoms appear?
Yes No
C. Facility Design Compliance
• Proposed Containment Level (check one). CL2
CL2 with extra precautions - Identify extra precautions: CL2 + RG3
• All work will be conducted at the proposed Containment Level Facility Design
requirements as per the Canadian Biosafety Standard and U of M Biosafety Manual? Yes No
D. Operational Practices Risk Assessment
• Will sharps be used? Yes No
• If YES, are you using safety engineered sharps? Yes No o If not, explain:
• If YES, do you have approved sharps containers (autoclavable, puncture resistant, secure lid) available for disposal? Yes No
• How and at what stage of the experiment is the cells inactivated or lysed? OR What is the method of terminal inactivation?
• Will all work be done in a BSC? Yes No
• If bench work is done, identify which procedures are being done
• Describe measures to be used for the protection of personnel and the environment.
• Will your experiments involve centrifugation? If yes, are sealed rotors, or sealed centrifuge safety cups available for use? Yes No
• Will you open the tubes in the BSC after centrifuging? Yes No • Identify any additional CL2 Operational Practices or safe work practices required:
• Largest volume used: < 1Litres 1-10LItres >10Litres
• Typical Volumes, containers, concentrations used:
• Disinfectants and decontaminants in use:
