Prevention, Diagnosis and Eradication of Mycoplasma Contamination in Cell Culture

¹ Beijing Key Laboratory of Emerging Infectious Diseases, Institute of Infectious Diseases, Beijing Ditan Hospital, Capital Medical University, Beijing 100015, P.R. China

² Beijing Institute of Infectious Diseases, Beijing, 100015, P.R. China.

³ National Center for Infectious Diseases, Beijing Ditan Hospital, Capital Medical University, Beijing 100015, P.R. China

⁴ National Key Laboratory of Intelligent Tracking and Forecasting for Infectious Diseases, Beijing,100015, P.R. China.

⁵ Department of Urology, Beijing Shijitan Hospital, Capital Medical University, Beijing, 100038, P.R. China.

⁶ State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100080, P.R. China

⁷ Shandong First Medical University & Shandong Academy of Medical Sciences, Shandong, 250000, P.R. China.

JBM 2023 , 10(1), 1; https://doi.org/10.14440/jbm.2023.407

Published: 1 November 2023

© 2023 by the author. Licensee POL Scientific, USA. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution 4.0 International License ( https://creativecommons.org/licenses/by/4.0/ )

Abstract

Mycoplasmas, which are the smallest and simplest prokaryotes, lack a cell wall but possess the ability to undergo self-replication. Mycoplasma contamination is a common problem for laboratories engaging in cell culture. Due to their small size, Mycoplasmas can easily permeate filters designed to prevent bacterial and fungal contamination in cell culture. Although Mycoplasma contamination usually does not result in cell death, it can significantly affect cell proliferation, metabolism, and cause chromosomal aberrations. Therefore, it is crucial to detect and eliminate Mycoplasma contamination in cell culture. This step-by-step protocol presents a comprehensive approach to prevent Mycoplasma contamination in cell culture, as well as to detect and eradicate Mycoplasma to ensure accurate experimental and sequencing results.

Keywords

Mycoplasmas

contamination

diagnostic

eradication

cell culture

References

1. Deeney AS, Collins R, Ridley AM. Identification of Mycoplasma species and related organisms from ruminants in England and Wales during 2005-2019. BMC Vet Res. 2021;17(1):325. Epub 2021/10/14. https://doi.org/10.1186/s12917-021-03037-y PMID: 34641885 PMCID: PMC8513359.
2. Pieters MG, Maes DJDos. Mycoplasmosis. public In: Jeffrey J. Zimmerman, Locke A. Karriker, Alejandro Ramirez, Kent J. Schwartz, Gregory W. Stevenson, Jianqiang Zhang, editors. Mycoplasmas in Swine. 2019. pp.863-83.
3. Malik, P., Mukherjee, S., Mukherjee, T.K. Microbial Contamination of Mammalian Cell Culture. public In: Mukherjee, T.K., Malik, P., Mukherjee, S. editors. Practical Approach to Mammalian Cell and Organ Culture. Springer, Singapore.2023. pp. 187-231.
4. Rottem S, Naot Y. Subversion and exploitation of host cells by mycoplasmas. Trends in microbiology. 1998;6(11):436-40. Epub 1998/12/10. https://doi.org/10.1016/s0966-842x(98)01358-4 PMID: 9846360.
5. Gill P, Pan J. Inhibition of cell division in L5178Y cells by arginine-degrading mycoplasmas: the role of arginine deiminase. Canadian journal of microbiology. 1970;16(6):415-9. Epub 1970/06/01. https://doi.org/10.1139/m70-071 PMID: 5423277.
6. Paddenberg R, Weber A, Wulf S, Mannherz HG. Mycoplasma nucleases able to induce internucleosomal DNA degradation in cultured cells possess many characteristics of eukaryotic apoptotic nucleases. Cell Death Differ. 1998;5(6):517-28. Epub 1999/04/14. https://doi.org/10.1038/sj.cdd.4400380 PMID: 10200503.
7. Olarerin-George AO, Hogenesch JB. Assessing the prevalence of mycoplasma contamination in cell culture via a survey of NCBI's RNA-seq archive. Nucleic Acids Res. 2015;43(5):2535-42. Epub 2015/02/26. https://doi.org/10.1093/nar/gkv136 PMID: 25712092 PMCID: PMC4357728.
8. Feng N, Huang X, Jia Y. Mycoplasma contamination affects cell characteristics and decreases the sensitivity of BV2 microglia to LPS stimulation. Cytotechnology. 2019;71(2):623-34. Epub 2019/04/05. https://doi.org/10.1007/s10616-019-00311-8 PMID: 30945036 PMCID: PMC6465415.
9. Vaisid T, Kosower NS. Calpastatin is upregulated in non-immune neuronal cells via toll-like receptor 2 (TLR2) pathways by lipid-containing agonists. Biochim Biophys Acta. 2013;1833(10):2369-77. Epub 2013/06/19. doi: 10.1016/j.bbamcr.2013.06.006. PubMed PMID: 23773963.
10. Hopfe M, Deenen R, Degrandi D, Köhrer K, Henrich B. Host cell responses to persistent mycoplasmas--different stages in infection of HeLa cells with Mycoplasma hominis. PLoS One. 2013;8(1):e54219. Epub 2013/01/18. https://doi.org/10.1371/journal.pone.0054219 PMID: 23326599 PMCID: PMC3543322.
11. Tsompana M, Buck MJ. Chromatin accessibility: a window into the genome. Epigenetics Chromatin. 2014;7(1):33. Epub 2014/12/05. https://doi.org/10.1186/1756-8935-7-33 PMID: 25473421 PMCID: PMC4253006.
12. Nikfarjam L, Farzaneh P. Prevention and detection of Mycoplasma contamination in cell culture. Cell journal. 2012;13(4):203-12. Epub 2012/01/01. PMID: 23508237 PMCID: PMC3584481.
13. Mukherjee S, Malik P, Mukherjee TK. Establishment of a Cell Culture Laboratory. public In: Mukherjee, T.K., Malik, P., Mukherjee, S., editors. Practical Approach to Mammalian Cell and Organ Culture. Springer, Singapore. 2023. pp. 43-82
14. Carrillo-Avila JA, de la Fuente A, Aguilar-Quesada R, Ligero G, Del Rio-Ortiz JM, Catalina P. Development and Evaluation of a New qPCR Assay for the Detection of Mycoplasma in Cell Cultures. Curr Issues Mol Biol. 2023;45(8):6903-15. Epub 2023/08/25. https://doi.org/ 10.3390/cimb45080435 PMID: 37623254 PMCID: PMC10453501.
15. Young L, Sung J, Stacey G, Masters JR. Detection of Mycoplasma in cell cultures. Nat Protoc. 2010;5(5):929-34. Epub 2010/05/01. https://doi.org/10.1038/nprot.2010.43 PMID: 20431538.
16. Benedetti F, Curreli S, Zella D. Mycoplasmas-Host Interaction: Mechanisms of Inflammation and Association with Cellular Transformation. Microorganisms. 2020;8(9). Epub 2020/09/10. https://doi.org/10.3390/microorganisms8091351 PMID: 32899663 PMCID: PMC7565387.

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