Mycoplasma: the insidious enemy Review
It’s the forbidden word in cell culture. It is every cell culturist’s nightmare, or at least it should be. Mycoplasma.
Cell culture contaminations of mycoplasma have been known for decades. The problem is serious, and with potentially 50% or more of cell-lines worldwide contaminated, mycoplasma can significantly impact the interpretation of biological results. To what extent and measures should we go to prevent or eliminate Mycoplasma contamination in cell culture?
Mycoplasma in the lab
Mycoplasma is a unique type of microorganism belonging to the class of Mollicutes. The first strains of mycoplasma were isolated at the Pasteur Institute in 1898, and to date 20 of the 190 or so species have been identified as bona fide contaminants in laboratory cell culture. Mycoplasmas are the smallest free-living organisms and considered the simplest of bacteria. Owing to their extremely basic genomes, mycoplasmas in fact are parasites exploiting host cells to fulfill their energy requirements and biosynthesis of their components.
Since first reported in the mid-1950s, mycoplasma contamination of culture cell lines has become a major problem in both basic research and in the production of biopharmaceuticals [1,2]. Mycoplasmas are structurally unlike other bacteria in that they lack a solid wall, instead possessing a “plasma”-like form. This, together with their minute size (~100 nm), means that mycoplasma contaminations go undetected for extended lengths of time and are resistant to a number of commonly used antibiotics, such as penicillin and streptomycin. Hundreds of mycoplasmas can attach to a single eukaryotic cell, eventually invading the host by fusing with the cell membrane. Upon entry into the cell, mycoplasmas multiply and circumvent defenses to survive, outnumbering host cells by 1000-fold. Contamination of a cell culture by mycoplasmas cannot be visualized by the naked eye or even by light microscopy and do not produce overt turbid growth commonly associated with bacterial and fungal contamination. The morphological cellular changes and growth rates of cell cultures can be minimal or unapparent.
Effects on cell culture
Given the high and potentially increasing incidence of mycoplasma contamination in cell lines worldwide, the issue cannot be ignored by any laboratory conducting cell culture as the manifestations are profound. Mycoplasma competes with host cells for biosynthetic precursors and nutrients and can alter DNA, RNA and protein synthesis, diminish amino acid and ATP levels, introduce chromosomal alterations and modify host-cell plasma membrane antigens.
A microarray analysis on contaminated cultured human cells revealed the extortionate effects that mycoplasma has on altering the expression of hundreds of genes including those encoding for receptors, ion channels, growth-factors, and oncogenes [3].
In addition, mycoplasmas exert significant effects on cultured immune cells such as monocytes and macrophages. Mycoplasmas contain highly immunogenic lipoproteins anchored on the outer face of the plasma membrane. These lipoproteins are recognized by specific pattern recognition receptors, in particular Toll-like receptor 2 (TLR2) present on immune cells. Upon recognition of the mycoplasmal lipoproteins, TLR2 induces the NF-kB pathway leading to the activation of these cells therefore biasing experimental results.
Get-rid off Mycoplasma in cell culture
There are three major sources leading to mycoplasma contamination of cell cultures: 1) incoming infected cells, 2) contaminated cell culture medium ingredients such as serum and trypsin 3) laboratory personnel infected with M. orale or M. fermentans. A contamination can spread rapidly to other cell lines through aerosol droplet
dispersion.
Once mycoplasmas have been detected, discarding the contaminated cell line remains the best recommended solution to eliminate and prevent spreading. In important cases, for instance to rescue a valuable cell line, an effective mycoplasma eradication treatment is needed. Some antibiotics that are selectively active on mycoplasma are currently available. Following treatment, they have been shown to eliminate mycoplasma and restore cell behavior and responses [5].
So the answer is clear and simple. All efforts must be taken to prevent or eliminate mycoplasma contamination in cell culture. Regular testing of cell cultures to ensure absence of mycoplasma in conjunction with good aseptic technique is strongly advised.
InvivoGen offers an easy and reliable mycoplasma detection method, PlasmoTest™. This cell-based assay exploits the ability of the immune system, in particular TLR2, to recognize mycoplasma and can be easily implemented as part of cell culture to routinely check for all types of mycoplasma contamination. Cell cultures found contaminated can be effectively treated with Plasmocin™ , a well-established antimycoplasma reagent, or Plasmocure™, an alternative mycoplasma removal agent, some of InvivoGen’s best sellers.
1. Robinson LB. et al., 1956. Contamination of human cell cultures by pleuropneumonia like organisms. Science. 124(3232):1147-8.
2. Armstrong SE. et al., 2010. The scope of mycoplasma contamination within the biopharmaceutical industry. Biologicals. 38(2):211-3.
3. Miller CJ. et al., 2003. Mycoplasma infection significantly alters microarray gene expression profiles. Biotechniques. 35(4):812-4.
4. Takeuchi O. et al., 2001. Discrimination of bacterial lipoproteins by Toll-like receptor 6. Int Immunol. 13(7):933-40.
5. Zakharova E. et al., 2010. Mycoplasma suppression of THP-1 Cell TLR responses is corrected with antibiotics. PLoS One. 5(3):e9900.
