4.4 Cell culture problems: identification and elimination

4.4.1    Cell cultures degenerate too quickly

This problem is more frequently encountered with cell cultures prepared in tubes than with flasks. Cell degeneration may lead to rounding up of cells and their detachment from the surface of the culture vessel. The most frequent reasons for rapid cell degeneration are: use of too high a seeding density, failure to change to MM at the proper time, or use of poor quality or too high concentration of fetal calf serum when preparing cultures. Cells rapidly divide, deplete available nutrients in the cell culture medium, and consequently produce acidic growth conditions and early cell death. The optimum cell count and growth conditions are likely to vary between cell lines and different batches of cells. It should be noted that cultures showing rapid cellular degeneration pose problems in poliovirus detection as they may show decreased sensitivity for virus isolation and degeneration may easily be mistaken for virus-induced cytopathic effects. The problem of cell degeneration can usually be overcome by appropriate adjustment of the cell count or fetal calf serum concentration. Laboratories may therefore need to establish those conditions that will allow cells to last for at least 5 to 7 days without degeneration.

4.4.2    Cell line cross-contamination

Cell cultures are grown in very rich media that can support the growth of a number of contaminants, from microbiological to other cell lines. Most fungal and bacterial contamination quickly overwhelms a culture and is usually visible to the naked eye within a short period of time. All cultures should be examined by eye and microscopically before being opened. Any cell culture with signs of contamination should be discarded without opening to minimize the chances of the contamination spreading to other cell cultures or media.

Whenever rapidly growing, continuous cell lines are maintained in a laboratory there is a risk of cell line cross-contamination. There is a long history of this problem, but it is often ignored. Cross-contamination of cell lines with very different susceptibilities for infection with different viruses (such as L20B and RD cells) will clearly affect interpretation of isolation results. If a laboratory discovers that non-polio enteroviruses grow readily in L20B cells, cell-line cross-contamination must be considered. Simple precautions must be taken to minimize the possibility of cross-contamination, including:

·         Only one cell line should be used in a BSC at any one time. After removal of the cell cultures from the cabinet, the cabinet should be swabbed down with a suitable disinfectant and the cabinet run for five minutes before introduction of another cell line.

·         Bottles or aliquots of medium should be dedicated for use with only one cell line.

·         Regularly return to frozen stocks — never grow a cell line for more than three months or 15 passages from stock passage level, whichever is the shorter period.

·         All culture vessels must be carefully and correctly labelled (including name of cell line, passage number and date of transfer), as must liquid nitrogen containers used for long-term cell storage.

If cross-contamination has occurred it can be readily identified by checking the virus susceptibility of the cells. For example L20B cells inoculated with a reference non-polio enterovirus, such as Coxsackie B, should show no cytopathic effect. RD cells have a characteristic mixed morphology and any change to this could indicate cross-contamination.

4.4.3    Mycoplasma contamination

Mycoplasma are small, self-replicating prokaryotes (0.3–0.8 μm diameter) that lack a cell wall and have the ability to cyto-absorb onto host cells. Contamination with Mycoplasma is usually not detected unless appropriate tests are carried out or some aspect of cell behaviour (e.g. ability to maintain monolayers) is observed to have changed. Mycoplasma contamination of cell lines is a very common problem, and laboratories that maintain cells through many passages but do not test for Mycoplasma probably have contaminated cell lines. Furthermore, there is a probability that all cell stocks in these laboratories are contaminated, as Mycoplasma spread readily among cell lines through reagents and media, the operator and the work surfaces. The origin of contamination can sometimes be traced back to Mycoplasma present in culture media supplements, such as bovine serum, or human oral Mycoplasma transferred by droplet infection during cell culture.

Mycoplasma contamination of propagated cell cultures is probably unavoidable but some precautions can minimize its occurrence, including:

·         Obtaining cell cultures from reputable suppliers, accompanied by documentation of Mycoplasma- negative status and method for testing. Reference laboratories that supply other laboratories with cell cultures must implement Mycoplasma testing. National laboratories need not implement Mycoplasma testing, once they follow the additional precautions provided below for handling cell cultures.

·         Adopting recommended quality control procedures to assure efficacy of sterilization methods for media and glassware.

·         Strict adherence to aseptic techniques in handling cultures.

·         Preparation of cell cultures within a BSC and minimizing traffic of personnel through work areas when cultures are prepared.

·         Avoiding conversation while cell cultures are being prepared.

·         Proper disinfecting of work surfaces when work is completed.

·         Adhering to recommendations for discarding and replacing cell cultures after every 15 passages.

·         Autoclaving before disposal of cell culture materials, and especially autoclaving of any cells found to be contaminated with Mycoplasma.

There are a number of methods for detecting Mycoplasma contaminants and these are summarized below.

Table 4.3: Summary of methods for detection of Mycoplasma

Cell cultures being checked for Mycoplasma should be cultured in antibiotic-free medium before being assayed as some antibiotics may suppress the growth of Mycoplasma.