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Mycoplasma: some basic facts
Contamination with Mycoplasma – Wikipedia, previously known as pleuropneumonia-like organisms or PPLO, is a common bane in cell cultures, with studies routinely finding many cell lines contaminated with it (1, 2).
Though 0.2 μm or even 0.1 μm pore size of sterilizing filters are normally recommended to filter culture media constituents such as FBS/FCS (Fetal Bovine/Calf Serum) to exclude mycoplasma as well as other micro-organisms (3), mycoplasma are extremely small, 300 to 800 nm in diameter, so filtering alone may be insufficient to exclude them (4, 5). Many mycoplasma strains are also unresponsive to commonly used cell culture antibiotics. ATCC, Bionique, BioReliance, ECACC, Mycoplasma Experience are companies that provide mycoplasma screening services (5). Unfortunately in vitro culture of eukaryotic cells appears to favor mycoplasma growth (6), especially given careless hands and lack of attention to detail.
Routine testing of cell lines for mycoplasma contamination is the only way to mitigate and minimize its harmful effect on biomedical research, something well-recognized as far back as 1994 (see below from 1).
‘100% of the cultures from labs without mycoplasma testing programs were contaminated, but only 2% of the cultures from labs that tested regularly.’
Mycoplasma: cell culture effects
Stealthy by nature and insidious in effect, mycoplasma contamination is much more difficult to discern unlike contamination by bacteria or fungi, whose effects are easily detectable through microscopy or pH (Phenol Red-containing culture media turn yellow through a rapid pH drop) or turbidity changes.
Old is often gold when it comes to cell culture resources and one of the best descriptions of effects of mycoplasma contamination on cell cultures is from a 1971 review (see below from 4)
‘Gross macroscopic changes in tissue cells infected with Mycoplasma sp. can range from inapparent or minimal unsuspected alterations to cytopathology and cell destruction reminiscent of viral infections. Macroscopic changes in morphology can be so minimal that they are not suspected even though a culture can reveal high titers of Mycoplasma. Cytopathie changes may be related to depletion of arginine in the medium by the Mycoplasma…Effects of cytopathic Mycoplasma sp. have been confused with virus infections…studies of cytopathic Mycoplasma strains have emphasized the risk involved in attributing all cytopathic effects to viruses…Many properties are shared by Mycoplasma and viruses: (a) filtrability: a number of strains of Mycoplasma (78) produced cells capable of passing through a 0.22 uL MIillipore filter, and it has been found (79) that the size of the smallest filtrable unit varies under different conditions of culture; (b) electron microscopic morphology: certain pleomorphic forms of rickettsiae, ornithosis virus, and Mycoplasma can be confused with one another (80); (c) sensitivity to ether (75); (d) ability to hemagglutinate (81, 82); (e) ability to cause hemadsorption (83); (f) resistance to some antibiotics; (g) inhibition of growth by antiserum (84, 85); and (h) induction of chromosomal aberrations (86, 87).’
A 1994 review (1) summarizes mycoplasma contamination effects on cell cultures as,
‘the ability to alter their host culture’s cell function, growth, metabolism, morphology, attachment, membranes, virus propagation and yield, interferon induction and yield, cause chromosomal aberrations and damage, and cytopathic effects including plaque formation ‘
Thus, mycoplasma contamination typically
- Affect the rate of cell proliferation (1, 4, 7).
- Induce morphological changes (4, 7, see below from 1).
- Cause chromosome aberrations (1, 7, see next one below from 8).
- Influence amino acid and nucleic acid metabolism (1, 7).
- Induce cell transformation (7).
- Yield poorly reproducible results from cell lines (9, 10).
- Activate primary immune cells (11).
- Change gene expression patterns (see next one below from 8), with one study (12) suggesting as many as 10% of gene expression studies including those published in leading science journals showed evidence of mycoplasma contamination.
Bibliography
1. Ryan, John A. Understanding and managing cell culture contamination. Corning Incorporated, 1994. https://pdfs.semanticscholar.org…
2. Uphoff, Cord C., and Hans G. Drexler. “Comparative PCR analysis for detection of mycoplasma infections in continuous cell lines.” In Vitro Cellular & Developmental Biology-Animal 38.2 (2002): 79-85. https://www.dkfz.de/gpcf/fileadm…
3. Clarke, Sue, and Janette Dillon. “The Cell Culture Laboratory.” Animal Cell Culture: Essential Methods (2011): 1-31.
4. Fogh, Jørgen, Nelda B. Holmgren, and Peter P. Ludovici. “A review of cell culture contaminations.” In vitro 7.1 (1971): 26-41. A review of cell culture contaminations
5. Davis, John M. “Basic techniques and media, the maintenance of cell lines, and safety.” Animal Cell Culture: Essential Methods (2011): 91-151.
6. Razin, Shmuel, and Leonard Hayflick. “Highlights of mycoplasma research—an historical perspective.” Biologicals 38.2 (2010): 183-190. https://www.researchgate.net/pro…
7. Thraves, Peter, and Cathy Rowe. “The Quality Control of Animal Cell Lines and the Prevention, Detection and Cure of Contamination.” Animal Cell Culture: Essential Methods (2011): 255-296.
8. Chernov, V. M., O. A. Chernova, and J. T. Sanchez-Vega. “Mycoplasma contamination of cell cultures: vesicular traffic in bacteria and control over infectious agents.” Acta Naturae (англоязычная версия) 6.3 (22) (2014). https://pdfs.semanticscholar.org…
9. Callaway, Ewen. “Contamination hits cell work: Mycoplasma infestations are widespread and costing laboratories millions of dollars in lost research.” Nature 511.7511 (2014): 518-519. https://www.nature.com/polopoly_…
10. Gedye, Craig, et al. “Mycoplasma infection alters cancer stem cell properties in vitro.” Stem Cell Reviews and Reports 12.1 (2016): 156-161.
11. Heidegger, Simon, et al. “Mycoplasma hyorhinis-contaminated cell lines activate primary innate immune cells via a protease-sensitive factor.” PloS one 10.11 (2015): e0142523. http://journals.plos.org/plosone…
12. Assessing the prevalence of mycoplasma contamination in cell culture via a survey of NCBI?s RNA-seq archive. https://pdfs.semanticscholar.org…