EVALUATION OF METHODS FOR THE DETECTION OF MYCOPLASMA CONTAMINATION OF CELL CULTURES AND THE EFFECTIVENESS OF ANTIMYCOPLASMA PREPARATIONS

This article presents the results of a study on the detection of mycoplasma infection in various cell lines and the evaluation of the effectiveness of antimycoplasma drugs. To detect mycoplasmas in cell cultures, DNA fluorochrome (DAPI, Apollo Scientific) staining methods, PlasmoTest™ (Invivogen, France), MycoStrip (Invivogen, France) and EZ-PCR™ (Biological Industries, Israel) kits were used, which are widely recommended for detecting mycoplasmas in cell culture. Based on the obtained research results, it was found that the antimycoplasma drugs MycoZap (Lonza Bioscience), Plasmocin (Invivogen, France), MRA (BIO-RAD) and ВМ-Cyclin (Roche) are effective in combating mycoplasma contamination of cell cultures. The method of detecting mycoplasmas with DNA fluorochromes, determining the presence of mycoplasma may be subjective, since its sensitivity to mycoplasmas is low. In turn, the MycoStrip set can give a false negative result due to the presence of mycoplasmas not included in the list of detectable species of this set. The positive and negative results of PlasmoTest were precisely correlated with the results of EZ-PCR. If mycoplasma contamination is suspected, DNA staining and the MycoStrip kit can be used for preliminary analysis, and PlasmoTest™ and EZ-PCR final confirmation.

1. Degeling, M. H., Maguire, C. A., Bovenberg, M. S., & Tannous, B. A. (2012). Sensitive assay for mycoplasma detection in mammalian cell culture. Analytical Chemistry, 84(9), 4227–4232. https://doi.org/10.1021/ac2033112.

2. Dvorakova, H., Valicek, L., & Reichelova, M. (2005). Detection of mycoplasma contamination in cell cultures and bovine sera. Journal of Veterinary Medicine, 50(6), 262–268. https://doi.org/10.17221/5622-VETMED.

3. Dobrovolny, P. L., & Bess, D. (2011). Optimized PCR-based detection of mycoplasma. Journal of Visualized Experiments, (52). https://doi.org/10.3791/2772.

4. Uphoff, C. C., & Drexler, H. G. (2002). Comparative PCR analysis for detection of mycoplasma infections in continuous cell lines. In Vitro Cell Developmental Biology, 38(2), 79–85. https://doi.org/10.1290/1071-2690(2002)038<0079:CPAFDO>2.0.CO;2.

5. Uphoff, C. C., & Drexler, H. G. (2004). Detecting mycoplasma contamination in cell cultures by polymerase chain reaction. Methods in Molecular Medicine, 88, 319–326.

6. Drexler, H. G., & Uphoff, C. C. (2000). Contamination of cell cultures Mycoplasma. In The Encyclopedia of Cell Technology (pp. 609–627). New York: Wiley.

7. Freshney, R. I. (2010). Culture of Animal Cells (6th ed.). Hoboken, NJ, USA: John Wiley & Sons.

8. Uphoff, C. C., & Drexler, H. G. (2010). Mycoplasma contamination of cell cultures. In M. C. Flickinger (Ed.), Encyclopedia of Industrial Biotechnology: Bioprocess, Bioseparation, and Cell Technology (pp. 3611–3630). New York, NY, USA: John Wiley & Sons.

9. Drexler, H. G., & Uphoff, C. C. (2002). Mycoplasma contamination of cell cultures: Incidence, sources, effects, detection, elimination, prevention. Cytotechnology, 39(2), 75–90.

10. СОП-GMP-НИИПББ-ПР-265-2020. ПЦР анализ по входному контролю культуры клеток на наличие микоплазмы с помощью набора EZ-PCR™ Mycoplasma Detection Kit.

11. Исмагамбетова, Д. Ж., Наханов, А. К., & Жаппарова, Г. А. (2014). Оценка влияния фармазина на элиминацию микоплазм в культурах клеток. В Инновационное развитие науки в обеспечении биологической безопасности (pp. 109). Гвардейский.

12. Ligasová, A., Vydržalová, M., Buriánová, R., Brůčková, L., Večeřová, R., Janošťáková, A., & Koberna, K. (2019). A new sensitive method for the detection of mycoplasmas using fluorescence microscopy. Cells, 8(12), 1510. https://doi.org/10.3390/cells8121510.

13. Young, L., Sung, J., Stacey, G., et al. (2010). Detection of mycoplasma in cell cultures. Nature Protocols, 5(7), 929–934. https://doi.org/10.1038/nprot.2010.43.

14. InvivoGen. (n.d.). Japanese cell bank study: Mycoplasma detection methods. Retrieved from https://www.invivogen.com/sites/default/files/pictures/japanese-cell%20bank-study–mycoplasma-detection-methods.pdf.

15. Armstrong, S. E., Mariano, J. A., & Lundin, D. J. (2010). The scope of mycoplasma contamination within the biopharmaceutical industry. Biologicals, 38(2), 211–213. http://dx.doi.org/10.1016/j.biologicals.2010.03.002.