What are the consequences of mycoplasma contamination?

Since mycoplasmas do not always appear to visibly harm the cell cultures they infect, why should researchers be concerned with using cultures infected with mycoplasma?  Because of their typically high concentrations in cell cultures, mycoplasmas can often out compete with the host cells for essential nutrients resulting in altered growth and production of proteins.  After all, the number of mycoplasma in a contaminated culture can easily outnumber the cells by a factor of 1000:1 or more (Rottem and Barile, 1993).  Usually there is an absence of visible morphological changes but the invisible effects on the cell cultures are what make mycoplasma contamination so serious a problem.  Mycoplasmas have been shown to alter virtually every cell culture property and characteristic measured (Table 2a; Barile, 1977; Del Giudice and Gardella, 1984; Rottem and Barile, 1993; Lincoln and Gabridge, 1998; Drexler and Uphoff, 2002).  In essence, the cells no longer behave the way they should.  Some of the effects are due to the removal of key medium components by the mycoplasmas leading to diminished ATP levels, cytotoxicity and culture starvation.  As potential human pathogens, they pose biosafety concerns as well as adversely affecting the quality and quantity of cell-based biopharmaceuticals, such as vaccines, interferons, monoclonal antibodies, recombinant proteins and other related therapeutics.  US regulatory agencies and international pharmacopoeias provide guidance and regulations concerning mycoplasma testing of master cell banks, working cell banks, virus seed lots, virus harvests and all licensed final lots of biologicals produced by cells (FDA Points to Consider in the Characterization of Cell Lines Used to Produce Biologicals, 1993; FDA 21 CFR 610.30 Subpart D Tests for Mycoplasma, 1973; European Pharmacopoeia (Ph. Eur.) 6.0 Vol. 1  Mycoplasmas, general chapter 2.6.7. Strasboug, France: Council of Europe; 2007; Japanese Pharmacopoeia Fifteenth Edition chapter 14:  Mycoplasma Testing for Cell Substrates Used for the Production of Biotechnological/ Biological Products; 2006).

Furthermore, the presence of mycoplasma in cell cultures directly impacts your work (Table 2b).  Using mycoplasma contaminated cultures in experiments calls into question the validity and significance of any research data generated.  Maintaining cell integrity is especially important within the expanding field of cell based assays and 3-D tissue models used for evaluating biological processes and product efficacy and safety.  The discoveries derived from these in vitro customized assays must be reliable and reproducible.  Another leading concern is the scientific publication of erroneous or misleading experimental results based on mycoplasma positive cultures.  Indeed, it is essential that mycoplasma testing be performed on those research cell cultures prior to submission for publication to ensure the validity of study results. In addition, one must also consider the financial impact of cleaning up contaminated cultures, obtaining fresh cultures, repeating experiments and interrupting production of biopharmaceuticals.  Finally, professional embarrassment may emerge with sharing contaminated cultures with colleagues or other researchers.

As illustrated, the existing data on the effects of mycoplasma contamination underscores the need to establish proper controls and testing regimens for cell cultures used in biomedical science.

The next section outlines why mycoplasma contamination rates are so high.

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Table 2a. Some of the many culture parameters affected or altered by mycoplasma contamination
Cell metabolism
  • DNA, RNA and protein synthesis
  • Uptake of medium components especially amino acids and nucleic acid precursors
  • ATP levels
Cell performance
  • Enzyme activity and expression
  • Growth rate, viability and plating efficiency
  • Membrane composition and receptor expression, binding and signal transduction
  • Nucleic acid synthesis leading to aneuploidy and other chromosomal aberrations
  • Macrophage and lymphocyte activation and responses
  • Interferon production and activity
Viral activity
  • Infectivity
  • Propagation and yields


Table 2b. Consequences of mycoplasma contamination on your work
 1. Loss of time, money and effort
 2. Loss of valuable cell and products
 3. Erroneous data and misleading publications
 4. Personal embarrassment
 5. Biosafety concerns
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