What?

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

Adherent cell lines will grow in vitro until they have covered the surface area available or the medium is depleted of nutrients. At this point the cell lines should be sub-cultured in order to prevent the culture dying. To subculture the cells they need to be brought into suspension. The degree of adhesion varies from cell line to cell line but in the majority of cases proteases, e.g. trypsin, are used to release the cells from the flask. However, this may not be appropriate for some lines where exposure to proteases is harmful or where the enzymes used remove membrane markers/receptors of interest. In these cases cells should be brought into suspension into a small volume of medium mechanically with the aid of cell scrapers.

Schematic diagram of "Subculture of Adherent Cell Lines"

Materials

• Media– pre-warmed to 37oC (refer to the ECACC Cell Line Data Sheet for the correct medium)
• 70% ethanol in water
• PBS without Ca2+/Mg2+
• 0.25% trypsin/EDTA in HBSS, without Ca2+/Mg2+
• Trypsin
• ELISA Plate
• Soybean trypsin Inhibitor

Equipment

• Personal protective equipment (sterile gloves, Laboratory coat, safety visor)
• Waterbath set to appropriate temperature
• Microbiological safety cabinet at appropriate containment level
• CO2 incubator
• Pre-labeled flasks
• Cell Culture Plates(96-well plate)
• Marker Pen
• Pipettes
• Ampule Rack
• Tissue

Procedure

1. View cultures using an inverted microscope to assess the degree of confluency and confirm the absence of bacterial and fungal contaminants.
2. Remove spent medium.
3. Wash the cell monolayer with PBS without Ca2+/Mg2+ using a volume equivalent to half the volume of culture medium. Repeat this wash step if the cells are known to adhere strongly.
4. Pipette trypsin/EDTA onto the washed cell monolayer using 1ml per 25cm2 of surface area. Rotate flask to cover the monolayer with trypsin. Decant the excess trypsin.
5. Return flask to the incubator and leave for 2-10 minutes.
6. Examine the cells using an inverted microscope to ensure that all the cells are detached and floating. The side of the flasks may be gently tapped to release any remaining attached cells.
7. Resuspend the cells in a small volume of fresh serum-containing medium to inactivate the trypsin. Remove 100-200uL and perform a cell count.
8. Transfer the required number of cells to a new labeled flask containing pre-warmed medium.
9. Incubate as appropriate for the cell line.
10. Repeat this process as demanded by the growth characteristics of the cell line.

Key Points

1. Some cultures whilst growing as attached lines adhere only lightly to the flask and 96 well plate, thus it is important to ensure that the culture medium is retained and the flasks are handled with care to prevent the cells detaching prematurely.
2. Although most cells will detach in the presence of trypsin alone the EDTA is added to enhance the activity of the enzyme.
3. Trypsin is inactivated in the presence of serum. Therefore, it is essential to remove all traces of serum from the culture medium by washing the monolayer of cells with PBS without Ca2+/Mg2+.
4. Cells should only be exposed to trypsin/EDTA long enough to detach cells. Prolonged exposure could damage surface receptors.
5. Trypsin should be neutralized with serum prior to seeding cells into new flasks otherwise cells will not attach.
6. Trypsin may also be neutralized by the addition of soybean trypsin inhibitor, where an equal volume of inhibitor at a concentration of 1mg/ml is added to the trypsinised cells. The cells are then centrifuged, resuspended in fresh culture medium and counted as above. This is especially necessary for serum-free cell culture plates.
7. If a CO2 incubator is not available gas the flasks for 1-2min with 5% CO2 in 95% air filtered through a 0.25m filter.

Source: Sigmaaldrich

Many cultures obtained from a culture collection, such as ECACC, will arrive frozen and in order to use them the cells must be thawed and put into culture. It is vital to thaw cells correctly in order to maintain the viability of the culture and enable the culture to recover more quickly. Some cryoprotectants, such as DMSO, are toxic above 4oC therefore it is essential that cultures are thawed quickly and diluted in culture medium to minimize the toxic effects.

A schematic diagram of "Resuscitation of Frozen Cell Lines"

Materials

• Media– pre-warmed to the appropriate temperature (refer to the ECACC Cell Line Data Sheet for the correct medium and size of flask to resuscitation into.)
• 70% ethanol in water
• DMSO

Equipment

• Personal protective equipment (sterile gloves, Laboratory coat, safety visor)
• Waterbath set to appropriate temperature
• Glass Bottom Dishes
• Microbiological safety cabinet at appropriate containment level
• CO2 incubator
• Pre labeled flasks
• Marker Pen
• Pipettes
• ELISA plates
• Ampule Rack
• Tissue

Procedure

1. Read Technical data sheet to establish specific requirements for your cell line.
2. Prepare the flasks as appropriate (information on technical data sheet). Label with cell line name, passage number and date.
3. Collect ampule of cells from liquid nitrogen storage wearing appropriate protective equipment and transfer to laboratory in a sealed container.
4. Still wearing protective clothing, remove ampule from container and place in a waterbath at an appropriate temperature for your cell line e.g. 37oC for mammalian cells. Submerge only the lower half of the ampule. Allow to thaw until a small amount of ice remains in the vial - usually 1-2 minutes. Transfer to class II safety cabinet.
5. Wipe the outside of the ampule with a tissue moistened (not excessively) with 70% alcohol hold tissue over ampule to loosen lid.
6. Slowly, dropwise, pipette cells into pre-warmed growth medium to dilute out the DMSO (cell culture flasks prepared in Step 2).
7. Incubate at the appropriate temperature for species and appropriate concentration of CO2 in atmosphere.
8. Examine cells microscopically (phase contrast) after 24 hours and sub-culture as necessary.

Key Points

1. Most text books recommend washing the thawed cells in media to remove the cryoprotectant. This is only necessary if the cryoprotectant is known to have an adverse effect on the cells. In such cases the cells should be washed in media before being added to their final culture flasks. See Protocol 7 for further details.
2. Do not use an incubator to thaw cell cultures since the rate of thawing achieved is too slow resulting in a loss of viability.
3. If a CO2 incubator is not available gas the flasks for 1-2 minutes with 5% CO2 in 95% air filtered through a 0.25m filter.
4. For some cultures it is necessary to subculture before confluence is reached in order to maintain their characteristics e.g. the contact inhibition of NIH 3T3 (Prod. No. 93061524) cells is lost if they are allowed to reach confluence repeatedly.

Scouce: ECACC Handbook Protocol 2

Since it was established in 1981, the Cell Culture Facility has made it easier, more economical and more effective to use mammalian cell cultures in research at Fox Chase. Cell culture procedures provide the cellular materials for many of the molecular methods used to study normal and cancer cell proliferation, cellular regulatory mechanisms, and normal and abnormal development. As a result, cell culture has become a core technique in current molecular and cell biological research.

Given below are a few of the essential "do’s and don'ts" of cell culture. Some of these are mandatory e.g. use of personal protective equipment (PPE). Many of them are common sense and apply to all laboratory areas e.g. cell culture plate. However some of them are specific to tissue culture.

The Do’s

1. Use personal protective equipment, (laboratory coat/gown, gloves and eye protection) at all times. In addition, thermally insulated gloves, full-face visor and splash-proof apron should be worn when handling liquid nitrogen.
2. Always use disposable caps to cover hair.
3. Wear dedicated PPE for tissue culture facility and keep separate from PPE worn in the general laboratory environment. The use of different colored gowns or laboratory coats makes this easier to enforce.
4. Keep all work surfaces free of clutter.
5. Correctly label reagents including flasks, medium and ampules with contents and date of preparation.
6. Only handle one cell line at a time. This common-sense point will reduce the possibility of cross contamination by mislabeling etc. It will also reduce the spread of bacteria and mycoplasma by the generation of aerosols across numerous opened media bottles and flasks in the cabinet.
7. Clean the work surfaces with a suitable disinfectant (e.g. 70% ethanol) between operations and allow a minimum of 15 minutes between handling different cell lines.
8. Wherever possible maintain separate bottles of media for each cell line in cultivation.
9. Examine cultures and media daily for evidence of gross bacterial or fungal contamination. This includes medium that has been purchased commercially.
10. Quality Control all media and reagents prior to use.
11. Keep cardboard packaging to a minimum in all cell culture areas.
12. Ensure that incubators, cabinet, centrifuges and microscopes are cleaned and serviced at regular intervals.
13. Test cells for mycoplasma on a regular basis.

The Don’ts

1. Do not continuously use antibiotics in culture medium as this will inevitably lead to the appearance of antibiotic resistant strains and may render a cell line useless for commercial purposes.
2. Don’t allow waste to accumulate particularly within the microbiological safety cabinet or in the incubators.
3. Don't have too many people in the lab at any one time.
4. Don't handle cells from unauthenticated sources in the main cell culture suite. They should be handled in quarantine until quality control checks are complete.
5. Avoid keeping cell lines continually in culture without returning to frozen stock.
6. Avoid cell culture becoming fully confluent. Always sub-culture at 70-80% confluency or as advised on ECACC's cell culture data sheet.
7. Do not allow media to go out of date. Shelf life is only 6 weeks at +4oC once glutamine and serum is added.
8. Avoid water baths from becoming dirty by using Sigma Clean (Prod. No.S5525).
9. Don’t allow essential equipment to become out of calibration. Ensure microbiological safety cabinets are tested regularly.
10. Anyway, Don't break the Cell Culture Plates(6 well cell culture plate, 24 well cell culture plate, 96 well cell culture plate).

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