Mycoplasma Contamination in Cell Culture

What are Mycoplasmas?

Mycoplasmas (class Mollicutes) are the smallest and simplest self-replicating organisms, belonging to bacteria, but devoid of a cell wall. Mycoplasmas are named because of its mycelated fungi-like structure with a flowering plasma-like structure. Their absence of cell wall and flexible membrane result in taking different shapes and consequently difficulties in identifying even under a high powered electron microscope. The small size of mycoplasma (0.15-0.3μm) is the main reason for their escape through filtering systems and also their growth in high concentration in mammalian cell cultures without any turbidity or other obvious symptom.

Introduction of Mycoplasma Contamination

Contamination of cell cultures with mycoplasma was first noted in the 1950s but is still regrettably often disregarded. Different from bacterial and fungal contaminations in cell culture, which are usually obvious and easily detected, mycoplasmas have become probably the most prevalent and serious microbial contaminant of cell culture systems used in research and industry today. Mycoplasmas are resistant to commonly used antibiotics and they cannot be detected visually by turbidity of fluid or under the inverted microscope. It is estimated that about 5 to 30% of the world's cell lines are contaminated with mycoplasmas. However, the actual rates are probably higher in a significant number of laboratories. It was reported that the level of mycoplasma contamination of the cell cultures evaluated in Mexico was 88.7%. Although >20 different species of mycoplasma have been isolated from cell cultures, >95% of infections are caused by six prevalent species, which are the following: M. arginini, M. fermentans, M. hominis, M.hyorhinis, M. orale and Acholeplasma laidlawii.

Signs of Mycoplasma Contamination in Cell Culture

The following symptoms may indicate mycoplasma contamination:

1. A once vigorous cell line grows poorly.

2. An adherent cell line is less adherent.

3. Cell line cannot be grown to high density (>1 × 106 cells/ml).

4. B or T hybridomas fail to be generated from cell fusion techniques.

5. Cell lines cannot be cloned by limiting dilution.

6. Large amount of debris is evident in the culture supernatant.

Mycoplasma contamination can interfere in bioassays. The following effects may indicate mycoplasma contamination:

1. Any inhibitory effect of monoclonal antibody or culture supernatant on any proliferation assay.

2. Any costimulatory effect of culture supernatants on B cell proliferation.

3. Cell lines with higher than expected [3H]thymidine incorporation.

4. Failure of T cell clones or hybridomas to give usual proliferation or lymphokine responses.

Effects of Mycoplasma Contamination in Cell Culture

The impacts of mycoplasma contamination on cell cultures are 1. inhibition of proliferation, 2. increment in cell death, 3. fragmentation of DNA and 4) morphological features of apoptosis, such as chromosomal aberrations, change of gene expression patterns, changes in cell membrane antigenicity, inhibition of cell metabolism, etc. The cytocidal effect leads to the production of non-lipid associated protein fraction. The presence of mycoplasmas in cell cultures leads to different troubles to laboratories, including loss of time, money, valuable cells and misleading publications, besides personal embarrassment and biosafety concerns.

Sources of Mycoplasma Contamination in Cell Culture

Here is a summary of common sources of mycoplasma contamination in the laboratory:

(1) Cross-contamination from other mycoplasma-positive cell cultures.

(2) Laboratory equipment and work surfaces.

(3) Laboratory personnel (often via respiratory tract infections).

(4) Cell culture media, sera and reagents.

(5) The liquid phase of LN2 cryostorage vessels.

(6) Feeder cell cultures.

(7) Laboratory animals.

Detection of Mycoplasma Contamination in Cell Culture

A large number of simple, reliable, sensitive and specific tests are now available to detect mycoplasma contamination in cell culture as the below table shows.

To learn more details of mycoplasma detection methods

Elimination of Mycoplasma Contamination in Cell Culture

The first choice to eliminate the mycoplasma contamination completely is by autoclaving the infected cultures and replacing them with fresh stocks known to be mycoplasma-free. However, all too often the cell line is not replaceable with a mycoplasma-free aliquot and purging of mycoplasmas from such cultures is a necessity

Four general types of procedures have been used to eliminate mycoplasmas from infected cell cultures: physical, chemical, immunological and chemotherapeutic treatment as follows.

(1) Physical procedures:

·   Heat treatment

·   Filtration through microfilters

·   Induction of chromosomal or cell membrane damage with photosensitizing

(2) Chemical procedures:

·   Exposure to detergents

·   Washings with ether-chloroform

·   Treatment with methyl glycine buffer

·   Incubation with sodium polyanethol sulfonate

·   Culture in 6-methylpurine deoxyriboside

(3) Immunological procedures:

·   Co-cultivation with macrophages

·   In vivo passage through nude mice

·   Culture with specific anti-mycoplasma antisera

·   Exposure to complement

·   Cell cloning

(4) Chemotherapeutic procedures:

·   Antibiotic treatment in standard culture

·   Antibiotic treatment plus hyperimmune sera or cocultivation

·   with macrophages

·   Soft agar cultivation with antibiotics

Administration of antibiotics is by far the most common and efficient approach. TransSafe^™ Mycoplasma Elimination Reagent (TransMyco-1+2) (Cat. No. FM401) and TransSafe^™ Mycoplasma Elimination Reagent (TransMyco-3) (Cat. No. FM411) are both produced based on antibiotics with low cytotoxicity.

Prevention of Mycoplasma Contamination in Cell Culture

There are a variety of methods to prevent mycoplasma contamination: (1) Improve aseptic techniques and practices. Following good aseptic techniques can help to reduce the risk of mycoplasma contamination. (2) Avoid using mycoplasma contaminated cultures. It is suggested to supply cells from reliable cell banks. (3) Only use antibiotics responsibly. Continuous use of antibiotics for a long time in cell cultures not only is not helpful, but also can cause resistance problems. However, the use of antibiotics (Penicillin/ Streptomycin) for a short term (the first two weeks) in primary culture is vital. Since antibiotics are unstable in the medium, it is highly suggested to replace antibiotic containing medium with fresh medium every two or three days. (4) Discard or treat mycoplasma contaminated cells.  For valuable and rare cells, it is suggested to treat them to eliminate mycoplasma infection. Otherwise, it is recommended to discard mycoplasma contaminated cells. (5) Quarantine new cells of any origin. It is necessary to quarantine cells from other laboratories and check for mycoplasma contamination before using them for any purposes. (6) Reduce aerosol generation. Avoiding activities which result in making aerosols can help to prevent mycoplasma contamination in cell cultures. TransGen has launched an antibiotic based product (TransSafe^™ Mycoplasma Prevention Reagent-Cat. No. FM501) with low cytotoxicity for convenient mycoplasma prevention.

References for Mycoplasma Contamination in Cell Culture

·   Drexler H G, Uphoff C C. Mycoplasma contamination of cell cultures: Incidence, sources, effects, detection, elimination, prevention[J]. Cytotechnology, 2002, 39(2): 75-90.

·   Nikfarjam L, Farzaneh P. Prevention and detection of Mycoplasma contamination in cell culture[J]. Cell Journal (Yakhteh), 2012, 13(4): 203.

·   Falagan-Lotsch P, Lopes T S, Ferreira N, et al. Performance of PCR-based and bioluminescent assays for mycoplasma detection[J]. Journal of microbiological methods, 2015, 118: 31-36.

·   Harlin H, Gajewski T F. Diagnosis and treatment of mycoplasma‐contaminated cell cultures[J]. Current protocols in cytometry, 2008, 43(1): A. 3C. 1-A. 3C. 7.

·   Geraghty R J, Capes-Davis A, Davis J M, et al. Guidelines for the use of cell lines in biomedical research[J]. British journal of cancer, 2014, 111(6): 1021-1046.

·   Chernov V M, Chernova O A, Sanchez-Vega J T. Mycoplasma contamination of cell cultures: vesicular traffic in bacteria and control over infectious agents[J]. Acta Naturae (англоязычная версия), 2014, 6(3 (22)).

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