Science

In general terms cultures derived from blood (e.g. lymphocytes) grow in suspension. Cells may grow as single cells or in clumps (e.g. EBV transformed lymphoblastoid cell lines). For these types of lines subculture by dilution is relatively easy. But for lines that grow in clumps it may be necessary to bring the cells into a single cell suspension by centrifugation and resuspension by pipetting in a smaller volume before counting.

Schematic diagram of "Subculture of Suspension Cell Lines"

Materials

• Media– pre-warmed to 37oC (refer to the ECACC Cell Line Data Sheet for the correct medium)
• 70% Ethanol in water

Equipment

• Personal protective equipment (sterile gloves, laboratory coat, safety visor)
• Waterbath set to 37oC
• Microbiological safety cabinet at appropriate containment level
• Elisa plates
• Centrifuge
• CO2 incubator
• Inverted phase contrast microscope
• Haemocytometer
• Pre-labeled flasks & Cell Culture Plates(6 Well Plate, 24 Well Plate, 96 Well Plate)

Procedure

1. View cultures using an inverted phase contrast microscope. Cells growing in exponential growth phase should be bright, round and refractile. Hybridomas may be very sticky and require a gentle knock to the flask to detach the cells. EBV transformed cells can grow in very large clumps that are very difficult to count and the center of the large clumps may be non-viable.
2. Do not centrifuge to subculture unless the pH of the medium is acidic (phenol red = yellow) which indicates the cells have overgrown and may not recover. If this is so, centrifuge at 150g for 5 minutes, re-seed at a slightly higher cell density and add 10- 20% of conditioned medium (supernatant) to the fresh media.
3. Take a small sample of the cells from the cell suspension (100-200uL - Protocol 6 - Cell Quantification). Calculate cells/ml and re-seed the desired number of cells into freshly prepared flasks without centrifugation just by diluting the cells. The data sheet will give the recommended seeding densities.
4. Repeat this every 2-3 days.

Key Points

1. If the cell line is a hybridoma or other cell line that produces a substance (e.g. recombinant protein or growth factor) of interest retain the spent media for analysis.

Source: Sigma-Aldrich

The aim of cryopreservation is to enable stocks of cells to be stored to prevent the need to have all cell lines in culture at all times. It is invaluable when dealing with cells of limited life span. The other main advantages of cryopreservation are:

• Reduced risk of microbial contamination
• Reduced risk of cross contamination with other cell lines
• Reduced risk of genetic drift and morphological changes
• Work conducted using cells at a consistent passage number
• Reduced costs (consumables and staff time)

There has been a large amount of developmental work undertaken to ensure successful cryopreservation and resuscitation of a wide variety of cell lines of different cell types. The basic principle of successful cryopreservation is a slow freeze and quick thaw. Although the precise requirement may vary with different cell lines as a general guide cells should be cooled at a rate of –1oC to –3oC per minute and thawed quickly by incubation in a 37oC waterbath for 3-5 minutes. If this and the additional points given below are followed then most cell lines should be cryopreserved successfully.

1. Cultures should be healthy with a viability of >90% and no signs of microbial contamination.
2. Cultures should be in log phase of growth (this can be achieved by using pre-confluent cultures i.e. cultures that are below their maximum cell density and by changing the culture medium 24 hours before freezing).
3. A high concentration of serum/protein (>20%) should be used. In many cases serum is used at 90%.
4. Use a cryoprotectant such as Cell Culture Plate(6 well plate, 24 well plate, 96 well plate) or glycerol to help protect the cells from rupture by the formation of ice crystals. The most commonly used cryoprotectant is DMSO at a final concentration of 10%, however, this is not appropriate for all cell lines e.g. HL60 where DMSO is used to induce differentiation. In such cases an alternative such as ELISA Plate should be used (refer to data sheet for details of the correct cryoprotectant). Sigma also offers ready-made cell freezing media containing DMSO , glycerol and a serum-free formulation containing DMSO.

Source: SIGMA-ALDRICH

Serum is a complex mix of albumins, growth factors and growth inhibitors and is probably one of the most important components of cell culture medium. The most commonly used serum is fetal bovine serum. Other types of serum are available including newborn calf serum and horse serum. The quality, type and concentration of serum can all affect the growth of cells and it is therefore important to screen batches of serum for their ability to support the growth of cells(take especial care of 96 well plate). In addition there are other tests that may be used to aid the selection of a batch of serum including cloning efficiency, plating efficiency and the preservation of cell characteristics.

Fetal bovine serum (FBS) has been used to prepare a number of biological and has an excellent record of safety. The recognition of Bovine spongiform encepalopathy (BSE) in 1986 and it’s subsequent spread into continental Europe along side the announcement of the probable link between BSE and a new variant of Creutzfeldt Jacob disease in Humans, stimulated an increased concern about safe sourcing of all bovine materials. In 1993 the Food and Drug Administration (FDA) "recommended against the use of bovine derived materials from cattle which have resided in, or originated from countries where BSE has been diagnosed. The current (European Union) EU guidelines on viral safety focus on sourcing, testing and paying particular attention to the potential risk of cross contamination during slaughtering or collection of the starting tissue. As far as BSE is concerned, the EU guidelines on minimizing the risk of BSE transmission via medicinal products, CPMP/BWP/877/96, recommends the main measures to be implemented in order to establish the safety of bovine material versus the BSE risk. Again, similarly the focus is on geographical origin, the age of the animals, the breeding and slaughtering conditions, the tissue to be used and the conditions of it’s processing.

The use of FBS in production processes of medicinal products is acceptable provided good documentation on sourcing, age of the animals and testing for the absence of adventitious agents is submitted. All responsible suppliers of FBS for bio-pharmaceutical applications will provide such documentation.

Recent regulatory requirements in Europe stress the importance of justifying the use of material of bovine, caprine or ovine origin in the production of pharmaceutical products. Thus, although FBS has been used for many years in the production process of many medicinal products such as viral vaccines , recombinant DNA products and ELISA Plate, at present there is a justified trend to remove all material of animal origin from manufacturing processes. Sigma-Aldrich has recognized this growing trend and works closely with customers to optimize animal free media formulations to meet each customer’s cell culture requirements.

Similarly the FDA has similar guidelines when accepting regulatory submissions. The FDA regulates all medicinal products for Human use, such as therapeutics, vaccines ,diagnostics and Cell Culture Plates, and, usually, the United States Department Agriculture (USDA) are not involved.

More infomation from Sigma-Aldrich

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

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.

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(especially the solid waste, such as flasks, centrifuge tubes, contaminated golves etc). 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(such as 15ml Centrifuge Tube, 50ml Centrifuge Tube), 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

Source: Sigma-Aldrich

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:

1. Identifying and evaluating the risks.
2. 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:

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

• Poorly characterized mammalian cell lines.

• 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.
• Low quality Cell Culture Dishs, Flasks adn Plates.
• 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, especially the Cell Culture Dish(35mm Cell Culture Dish, 60mm Cell Culture Dish, 100mm Cell Culture Dish). 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.

Source: ECACC Handbook