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The Microscopic Cell Culture Plague: Mycoplasma

Mycoplasma is a class of bacteria that breaks the mold of typical bacterial classification – it does not have a cell wall. This feature makes it notoriously resistant to most common antibiotics like penicillin and cephalosporins, which are more effective on Gram-positive and Gram-negative cells (which have cell walls). Beyond being more resistant to go-to antibiotics, the fact that a cell wall is missing means that Mycoplasma cells have more fluidity and elasticity, which allows them significant malleability. Their small size (0.15 – 0.3 µm) and lack of a rigid outer wall enables them to ooze past 0.2 µm filters intended for sterile filtration through bacterial retention.

Mycoplasma poses threats to cell cultures that no other biological contaminant can. This is a challenge for both manufacturers and consumers of cell culture media. Cell culture is critical to the progression of research as well as to cell therapy manufacturing. The infection of a cell culture with Mycoplasma cells can severely affect the metabolic activity and the culture cells’ physiology, which ultimately results in lost time and money. Inaccurate data can also be attributed to Mycoplasma contamination. Biological sterility testing, as described in various pharmacopeias, usually detects aerobic and anaerobic microbial contamination caused by less unique Gram-positive and negative bacteria, most of which can be removed through sterile filtration.

Mycoplasma-free Cells
Fig.1 Mycoplasma-free Cells (Left) and Mycoplasma-contaminated Cells (Right)


Cell culture media must be evaluated using a different method, which is often time-consuming, especially if employing the compendial methods that require lengthy incubation. Real Time Polymerase Chain Reaction (RT-PCR or qPCR) methods can circumvent these lengthy and costly efforts to remain Mycoplasma free. RT-PCR methods also provide the advantage of being faster than traditional PCR methods and are an example of an effective and robust means for Mycoplasma detection than can be employed in lieu of lengthy compendial methods. RT-PCR based myco-detection methods have the ability to detect over a large variety of Mycoplasma species and are capable of excluding all other cellular DNA, whether prokaryotic or eukaryotic, to provide a means of specifically identifying the presence of Mycoplasma in cell cultures.

This highly specific method for rapid detection is a wave towards the future of bioproduct testing that will inevitably result in a shift in the amount of time and resources spent on the mollicute menace Mycoplasma. in general, the increased speed and accuracy of microbial testing is critical to the timely, safe, and successful development of advanced therapies.