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Contamination of Cell Cultures

Cell cultures may become contaminated by bacteria, molds, viruses and mycoplasmas. In general, contamination by bacteria and molds are easy to detect by visual inspection. Mycoplasmas, however, are perhaps the most severe and difficult cell culture contaminant to observe and handle. Since its discovery in 1956 (Armstrong et al. 2010), the presence of mycoplasmas in cell cultures has been a great challenge, both in research as well as in industrial laboratories and production facilities for cell-derived biological and pharmaceutical products. Although the risks and consequences of mycoplasma infection have been known for decades, recent studies (Drexler and Uphoff 2002) indicate that there has been no decline in infection rates. Mycoplasmas are small (0.1–0.6 μm) bacteria that lack cell wall. Thus, they are difficult to observe by conventional light microscopy. Furthermore, they are unaffected by antibiotics that target cell wall biosynthesis like penicillin and β-lactams, so they will continue to grow in infected cell cultures despite the presence of standard antibiotics and antimycotics. Mycoplasma species have been found associated with a range of hosts, including humans and other mammals, birds, reptiles, insects and plants. Despite the fact that more than 100 mycoplasma species have been described, most mycoplasmas that contaminate cell culture laboratories belong to only six species of bovine, swine and human origin. Bovine and swine mycoplasmas typically derive from contaminated sera or other animal-derived products used in the cell culture laboratory. Currently, many infections are transfered from already infected cultures within the same laboratory due to inadequate aseptic and cell culture techniques or from equipment that has been contaminated through prior handling of infected cells. However, laboratory personnel also represent an important source of mycoplasma infections, and mycoplasma species from humans, mainly of oral origin, are responsible for 40–80 % of mycoplasma infections in cell cultures. In total it is estimated that 15–35 % of all cultures based on continuous cell lines are mycoplasma infected (Barile et al. 1973), while contaminated cultures based on primary cells are seldom found.

Their flexibility in shape due to the lack of cell wall, allows most mycoplasmas to pass through 0.2 μm filters traditionally used for sterilization of media and medium ingredients. For the same reason they are difficult to observe in cell cultures. Moreover, despite the richness of standard culture media, mycoplasmas grow slowly, a fact that contributes to the problems with detecting them, visually or by change in turbidity. Although we tend to consider mycoplasmas mainly as a problem in cell culture work, it should be remembered that the mycoplasma family also includes a number of human pathogens.

How will mycoplasma infection affect the cultivated cells? Different mycoplasma species will introduce different problems, but in general, mycoplasma infections tend to influence a number of cellular processes leading to decreased growth rate due to inhibition of protein-, DNAand RNA synthesis. Additionally, they introduce changes in gene expression that can be measured as down-regulation of cytokine and growth factor secretion, expression of receptors, intracellular signaling molecules, ion channels etc.

How to avoid contamination by mycoplasmas? A few major guidelines should be followed: (1) Strict aseptic techniques are an absolute requirement. Minimize talking, and do not practice mouth pipetting when working with cell cultures. Never pour medium between bottles and flasks, and avoid crowding in the laminar flow hood. Unnecessary equipment in the hood will contribute to turbulence in the laminar airflow thus increasing the risk of contaminating flasks and culture plates. Avoid unnecessary traffic in the vicinity of the sterile hood. (2) All surfaces in laminar flow hoods, incubators and water baths should be regularly cleaned and disinfected (70 % ethanol or isopropanol). (3) Try to avoid unnecessary and excessive use of antibiotics. Routine work may preferably be carried out without addition of antibiotics to the medium. Use of antibiotics will camouflage poor aseptic techniques, and will contribute to worsen the problem. (4) When new cell lines arrive in the laboratory, they should be cultivated separated from other cell cultures, rigorously tested and found to be mycoplasma free before being admitted to the cell culture laboratory. (5) Establish a routine for frequent testing of the cultures. This is an absolute requirement for a responsible scientist. If a culture or cell line is found to be infected, cells, culture trays, medium bottles and any other equipment that has been in contact with the contaminated culture should be destroyed by autoclaving.

There are several ways to detect mycoplasma infection in cell cultures, but today, most people rely on PCR technique which is fast and sensitive. Results can be obtained in hours. Several vendors provide kits with specially designed primers that allow selective amplification of mycoplasma DNA for PCR-detection with a sensitivity that is close to the direct culture approach. Thus, for routine purposes PCR represents the method of choice.

Mycoplasma infection of cell lines and particularly clones of valuable primary cells can be a disaster. It may be possible to “cure” the cells with antibiotics. Typically, antibiotics like ciprofloxacin, minocyclin or a combination of tiamulin and minocyclin, have been demonstrated to be effective. However, toxic effects causing cell death is not uncommon. In general, cleaning up a contaminated culture should only be considered as an alternative for absolutely irreplaceable cultures provided that the source of the contamination has been identified and removed from the laboratory.

References

Armstrong SE, Mariano JA, Lundin DJ (2010) The scope of mycoplasma contamination within the biopharmaceutical industry. Biologicals 38:211–213

Barile MF, Hopps HE et al (1973) The identification and sources of mycoplasmas isolated from contaminated cell cultures. Ann N Y Acad Sci 225:251–264

Drexler HG, Uphoff CC (2002) Mycoplama contamination of cell cultures: Incidence, sources, effects, detection, elimination, prevention. Cytotechnology 39:75–90

Heeneman S, Deutz NE, Buurman WA (1993) The concentrations of glutamine and ammonia in commercially available cell culture media. J Immunol Methods 166:85–91

Roth E, Ollenschlager G, Hamilton G, Simmel A, Langer K, Fekl W, Jakesz R (1988) Influence of two glutamine-containing dipeptides on growth of mammalian cells. In Vitro Cell Dev Biol 24:696–698

 
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