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Contaminations

Mycoplasma Contamination

Mycoplasmas in Cell Cultures

Mycoplasma contaminations can affect virtually every cell culture parameter and may therefore lead to unreliable experiments and unreproducible or incorrect research results. Despite these severe consequences, the risk of mycoplasma contamination in cell cultures is often being underestimated. To assure reliable research data, testing for mycoplasma contamination is a necessary quality control procedure.

Several independent studies have shown that 15 -35% of all existing human and animal cell lines that are available worldwide are contaminated with mycoplasmas. This is a major concern in any public or commercial cell bank and in any experimental research set up exploiting cell cultures.

Mycoplasmas can be introduced into cell cultures through many different routes, such as

  • animal- or plant-derived media components (raw materials, e.g. serum, peptones)
  • laboratory personnel
  • tissue used to establish primary cell cultures
  • cross-infection from other contaminated cell cultures (the most common route!)

Although these sources of contamination can be largely eliminated through GMP and GLP precautions, they still need to be considered when investigating the source of a mycoplasma contamination.

Contaminated cultures can be treated with specific antibiotics or so-called mycoplasma "elimination" reagents. A complete clean-up is, however, practically impossible. This is at least partially due to the fact that some mycoplasma species or strains are capable to invade cultured eukaryotic cells, thereby circumventing "elimination" procedures. Internalized mycoplasmas in turn can leave the cells and contaminate new cells. The consequence: re-occurring mycoplasma contamination in previously reported "clean" cell cultures. Therefore, cell cultures tested positive for mycoplasmas should - if possible - be discarded to avoid any spread of contamination.

Whereas contamination of cell cultures by "classical" cell-wall containing bacteria or fungi cause visible effects, mycoplasmas are different. Neither an altered turbidity nor a change in the pH of cell culture media clearly signalizes a mycoplasma contamination. Furthermore, their extremely small cell size makes them impossible to detect with a standard bright-field or phase-contrast microscope.

Avoiding mycoplasma contamination in the first place should thus be highest priority, but is difficult to achieve. Due to the lack of a cell wall, mycoplasmas are highly plastic and pleomorphic and therefore pass easily through standard filter membranes used to sterilize cell culture reagents. Neither sterile filtration nor treatment with standard antibiotics used in cell biology can guarantee mycoplasma-free cell cultures. Some mycoplasma species or strains are even cell-invasive and may thus ‘hide’ inside cells during the treatment with antibiotics and other "decontamination" reagents. For these reasons mycoplasma contaminations can spread quickly, and their presence in cell cultures and bioreactors may go unnoticed for longer periods of time.

Basically, there is just one efficient safety precaution to maintain clean, mycoplasma-free cell cultures: the periodic testing for the possible presence of mycoplasmas. Only the commitment to routine mycoplasma testing by the conventional culture methods or newly available rapid NAT-based testing assays can minimize the risk of covert contamination that can lead to serious problems. Carrying out these test and interpreting the results requires solid training and experience and cannot always be done in-house. Outsourcing mycoplasma tests to Mycoplasma Biosafety is the alternative that brings along several advantages: unambiguous results in the shortest possible time, no requirement for the maintenance of an in-house mycoplasma testing facility, and free resources to concentrate on the core business to name just some of them.

 

Mycoplasmas in Biopharmaceutical Processes

Mycoplasma contamination of cell banks and virus stocks may cause serious problems in biotechnological and biopharmaceutical manufacturing. The international regulatory authorities have therefore published regulatory guidelines how to demonstrate that cell culture-derived products are free of mycoplasmas in order to ensure product safety, purity, and potency.

Mycoplasma contamination during production processes of biological medicinal products may result in decreased product quality and yields by affecting host cell metabolism and expression levels, inconsistency of manufacture, lost production times, and potential adverse effects in patients.

The most common mycoplasma species that contaminate cell cultures and which are also occasionally found in biotechnological and biopharmaceutical processes include Acholeplasma laidlawii, Mycoplasma argininiMycoplasma fermentans, Mycoplasma hyorhinis, Mycoplasma orale and Mycoplasma salivarium.
Regulatory authorities such as the EMA and the FDA therefore demand that biological products which are prepared in cell culture substrates and are intended for clinical use, need to be demonstrably free of mycoplasmas.
Safeguards at multiple levels of the production process assure that mycoplasmas do not contaminate production cultures or final products. Such safeguards include

  • demonstrating the absence of mycoplasmas in master/working cell banks and virus seed lots
  • in-process control testing for the potential presence of mycoplasmas in each bioreactor run

Mycoplasma Biosafety is specialized in regulatory mycoplasma testing according to various international regulatory protocols. You are welcome to contact us, if you have any questions regarding this topic.

 

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