Safety Aspects of Cell Culture

1.Risk Assessment

The main aim of risk assessment is to prevent injury, protect property and avoid harm to individuals and the environment. The performance of risk assessment is a legal requirement under the Health and Safety at Work Act, UK. There are other EC directives covering Health and Safety at Work, you can visit the European Agency for Safety and Health at Work website www.europe.osha.eu.int for information on legislation and standards, or you should contact your on-site representative. Consequently risk assessments must be undertaken prior to starting any activity. The assessment consists of 2 elements:

Identifying and evaluating the risks.

Defining ways of minimizing or avoiding the risk.

For animal cell culture the level of risk is dependent upon the cell line to be used and is based on whether the cell line is likely to cause harm to humans. The different classifications are given below:

Low risk: Non human/non primate continuous cell lines and some well characterized human diploid lines of finite lifespan (e.g. MRC-5).

Medium risk: Poorly characterized mammalian cell lines.

High risk: Cell lines derived from human/primate tissue or blood.

Cell lines with endogenous pathogens (the precise categorization is dependent upon the pathogen) – refer to ACDP guidelines, 1985, for details.

Cell lines used following experimental infection where the categorization is dependent upon the infecting agent - refer to ACDP guidelines, 1985, for details*.

*Advisory Committee on Dangerous Pathogens (1985) Categorization of Biological Agents According to Hazard and Categories of Containment, 4th edition, HSE books, Sudbury, UK

A culture collection, such as ECACC will recommend a minimum the containment level required for a given cell line based upon its risk assessment. For most cell lines the appropriate level of containment is Category 2. However, this may need to be increased to Category 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 HTLV Category 3 containment is required.

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 labeled and stored, are also important for reducing risk and making the laboratory a safe environment in which to work. Staff training and the use of written standard operating procedures and risk assessments will also reduce the potential for harm. Training courses covering the basics of tissue culture safety are offered by ECACC.

2.Disinfection

Methods designed for the disinfection/decontamination of culture waste, work surfaces and equipment represent important means for minimizing the risk of harm.

The major disinfectants fall into four groups and their relative merits can be summarized as follows:

Hypochlorites (e.g. Chloros, Presept)

*Good general purpose disinfectant

*Active against viruses

*Corrosive against metals and therefore should not be used on metal surfaces e.g. centrifuges

*Readily inactivated by organic matter and therefore should be made fresh daily

*Should be used at 1000ppm for general use surface disinfection, 2500ppm in discard waste pots for washing pipettes, and 10,000ppm for tissue culture waste and spillage

NB: When fumigating a cabinet or room using formaldehyde all the hypochlorites must first be removed as the two chemicals react together to produce carcinogenic products.

Phenolics (e.g. Sudol, Hycolin)

*Not active against viruses

*Remains active in the presence of organic matter

Alcohol (e.g. ethanol, isopropanol)

*Effective concentrations 70% for ethanol, 60-70% for isopropanol

*Their mode of activity is by dehydration and fixation

*Effective against bacteria. Ethanol is effective against most viruses but not nonenveloped viruses

*Isopropanol is not effective against viruses

Aldehydes (e.g. glutaraldehyde, formaldehyde)

* Aldehydes are irritants and their use should be limited due to problems of sensitization

*Glutaraldehyde may be used in situations where the use of hypochlorites is not suitable e.g. cleaning of centrifuge bowls or materials constructed of stainless steel that may be attacked or corroded by using hypochlorite solutions.

3.Waste Disposal

Any employer has a ‘duty of care’ to dispose of all biological waste safely in accordance with national legislative requirements. Given below is a list of ways in which tissue culture waste can be decontaminated and disposed of safely. One of the most important aspects of the management of all laboratory-generated waste is to dispose of waste regularly and not to allow the amounts to build up. The best approach is ‘little and often’. Different forms of waste require different treatment.

*Tissue culture waste (culture medium) - Inactivate overnight 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 autoclaved prior to incineration. These bags are available from Bibby Sterilin and Greiner.

*If at all possible waste should be incinerated rather than autoclaved.