Mycoplasma Contaminations of Cell Cultures
Mycoplasma Detection and Elimination
- Fig. 1: Percentages of false and mycoplasma-contaminated leukemia and lymphoma cell lines (ref. 2, updated December 2008)
- Fig. 2: Detection of mycoplasma contamination by FISH. Fluorescent micrograph of an adherently growing HELA cell line infected with M. fermentans. Cells were grown on slides, fixed with 4% paraformaldehyde and exposed to FITC-labeled oligonucleotides specific for mycoplasma (green). The cells were counterstained with TRITC-phalloidin to show the cytoplasma (red) and with DAPI to stain the nuclei of the eukaryotic cells (blue).
- Fig. 3: Detection of Mycoplasma Contamination by PCR. Ethidium bromide-stained gel containing the reaction products following PCR amplification. Products of about 510 bp represent the mycoplasma wild type and the products of about 1000 bp derive from the internal control DNA which was added to the reaction b. 1 and 12: 100 bp ladder; 2-8: different cell lines (sample 5 was inhibited by Taq polymerase inhibitors); 9: positive control; 10: internal control; 11: water control.
- Table 1: Antibiotics effective against mycoplasmas
- Dr. Cord C. Uphoff, DSMZ – German Collection of Microorganisms and Cell Cultures
- Prof. Dr. Hans G. Drexler, DSMZ – German Collection of Microorganisms and Cell Cultures
One group of bacteria regularly escapes routine inspections of cell cultures for contaminations: mycoplasmas. These organisms belong to the smallest known bacteria and their common distinguishing mark is the lack of a rigid cell wall. Their metabolic capabilities are highly reduced and to cover the requirements for nutritions and basic components of the metabolism, mycoplasmas live in close community with other higher organisms . The artificial environment of cell cultures provide optimal conditions for the growth of a number of mycoplasma strains, due to almost unlimited supply of nutrients and the absence of an immune system. Since the contaminations often remained undetected, mycoplasmas spread in cell cultures over the years and became the biggest problem in cell culture technology with more than 20% infection rate among continuous cell lines .
Although mycoplasmas can coexist with eukaryotic cells for years without apparent effects, they cannot be regarded as harmless sponger. Mycoplasmas can have considerable consequences on the cell culture, their products and obviously on the results of experimental studies. A number of investigations showed that beside diminished growth and decreased viability numerous biological activities of the eukaryotic cells are directly or indirectly affected. However, no general effects can be appointed, but the manifestation of the infection depends particularly on the infecting mycoplasma strain, the type of the cell culture and on the culture conditions . Freedom from mycoplasma infection is thus required for product safety as well as for the reliability of scientific studies. Periodic inspection of the cell culture for mycopalsma contaminations and effective eradication of the contaminants is required.
Nowadays already infected cell cultures in combination with negligence regarding handling of the cell cultures have to be regarded as main source of contamination for other cell cultures. There are several reasons to argue for this assumption: (a) cell culture supplements are usually tested for mycoplasmas or are treated with gamma irradiation; (b) the low infection rate of primary cell cultures (less than 1%); (c) the limited number of mainly seven in cell cultures occurring mycoplasma species (Mycoplasma arginini, M.
bovis, M. fermentans, M. hominis, M. hyorhinis, M. orale, and Acholeplasma laidlawii); (d) the finding that either none or all of the cell lines of a given laboratory are infected with a single mycoplasma strain; and (e) the comparably high rate of cell line cross contaminations (ca. 13%) (fig. 1). Thus, techniques which prevent spread of mycoplasmas should be strictly followed and the uncontrolled exchange of cell cultures should be avoided .
A number of methods were described for the detection of mycoplasmas in cell cultures, but only a few of them are suited for use in routine cell culture technology. In principle, two independent assays should be employed, since none of the tests are absolutely reliable.
One of the first assays - and still the standard assay regarding pharmaceutical and medical applications - was the microbiological culture method. An aliquot of the cell culture is inoculated into complex liquid medium and transferred after four days onto agar plates. Typical mycoplasma colonies usually develop after three to seven days. They often display a dense centre and a transparent corona ("fried eggs") under the inverted microscope. Even under optimal conditions, some highly fastidious strains do not grow on these media (e.g. M. hyorhinis). The assay is highly sensitive, but might take up to three weeks.
Another standard method mentioned in the European Pharmacopoeia is the staining with DNA binding fluorescence stains like DAPI (4,6-diamidino-2-phenylindole) or Hoechst 33258. This assay requires the culture of a mycoplasma negative indicator cell line (e.g. MDCK, Vero, NIH-3T3), which supports the growth of mycoplasmas very well. Supernatant of the cell culture to be tested is added to the indicator cell line, cultured for several days and subsequently stained with the fluorescent dye. Usually the detection is very reliable. The direct staining of the cell culture to be tested is only recommended as rapid periodic analysis for continuously cultured cell lines. Newly developed detection methods with mycoplasma-specific fluorescence-labeled probes (fluorescence in situ hybridization) can enhance the sensitivity and specificity and might overcome the need for an indicator cell line (fig. 2).
Several more convenient, but also sensitive detection assays were developed on the basis of molecular biological and biochemical methods to expedite the detection. The PCR assay belongs to the most sensitive analytical methods and several variations were described or are commercially available for mycoplasma detection. But this technique is also susceptible to errors leading to false results. False positive results are produced by reagents contaminated with mycoplasma or mycoplasmal DNA, whereas false negative results are often the result of the presence of Taq polymerase inhibitors. Thus, a DNA extraction method and the use of an internal control DNA for each sample is highly recommended (fig. 3). It is also necessary to optimize the PCR assay individually, as different Taq polymerases, buffers or thermal cyclers can have a strong impact on the sensitivity and specificity of the PCR . The DSMZ offers non-infectious DNAs as positive and internal controls for the establishment and optimization of the PCR assay.
Further efficient detection methods for mycoplasmas are DNA-RNA-hybridization assays with radioactive or acridinium ester labeled probes (Gen Probe), immunostaining with monoclonal antibodies, and the determination of ATP produced by mycoplasma specific enzymes (MycoAlert). Regarding the choice of specific detection assays, it is important that the test detects at least all the aforementioned mycoplasma species. Continuously cultured cells should be tested for contamination once every month.
Several methods were suggested to eradicate mycoplasmas from cell cultures. But experience showed that the employment of specific antibiotics is the most efficient and convenient way to overcome mycoplasma infections. The fact that mycoplasmas are peculiar organisms is also shown in the resistance of those bacteria against various antibiotics (particularly penicillin and streptomycin). They lack many of the points of attack of the antibiotics. Until now, three classes of antibiotics were shown to be effective against mycoplasmas: fluoroquinolones, macrolides and tetracyclines (table 1). The substances can also be applied alternating, but a simultaneous treatment with a quinolone and one of the other classes is not recommended. The curation rate for the antibiotics is 65-85%, depending on the antibiotic used . Not only resistances occur, but the antibiotics can also kill the eukaryotic cells if they are already massively affected by the mycoplasma contamination or react sensitive to the application of the antibiotic. Attention should be payed that the concentration of the antibiotic remains constant by frequent change of the medium and that the cell density is kept high during the treatment. The detection assay should be performed two weeks after the treatment at the earliest.
Antibiotics should not be applied prophylactically, because this might generate resistant clones and the growth of the mycoplasmas can be inhibited. Subliminal contaminations can occur and the infections might spread undetected over all cultures.
References are available from the author.