Avoiding contamination makes up a crucial objective of a cell culture lab. Some contaminants create bigger problems than others. Mycoplasma, for example, contaminates many cultures, ranging from 10 to 70% depending on the study. At any level, the keys to managing mycoplasma depend on detection, elimination, and control.
Recently, scientists at Houston Methodist Hospital wrote: “Mycoplasma cell line contamination is an intractable and serious problem in biological experiments. Due to their size, mycoplasma cannot be visualized and therefore, can affect the results of cell experiments in imperceptible ways, leading to erroneous or misleading results.” For an overview of the potential problem, read this blog post from Germany-based PromoCell.
Some experts see variations in mycoplasma contamination that can fluctuate with different cell lines. From mycoplasma services at Eurofins BioPharma Product Testing’s Pennsylvania location, for instance, group leader and principle microbiologist Christopher Smith said, “We have seen a significantly lower rate of contamination than the 10–70% range while testing cell lines for our biopharmaceutical industry clients, but we have seen contaminations of cell cultures from primary cell lines.” The lower rate of infection seen by Smith’s team “could be due to the extensive testing that goes into research and development of production cell lines for biopharmaceuticals, prior to a cell line being developed further for production,” he explained.
For a general idea of the concern over bacterial infections, I turned to PubMed. On June 12, 2o2o, I searched “mycoplasma AND ‘cell culture’”, which returned 468 articles—going back to 1956. That’s not a huge number of citations, but they are on the rise, with a high number of 30 in 2018. Even in 2020, scientists from the U.S. National Institutes of Health’s National Center for Advancing Translational Sciences wrote: “Two key pitfalls in tissue culture are 1) cell line authenticity and 2) mycoplasma contamination.” So, this is a problem that cell-culture scientists still face.
Scientists deal with mycoplasma in various ways, and available tools simplify that process.
Tools for detection
Scientists can test cultures for mycoplasma in different ways. The detection “can be accomplished using traditional compendial agar/broth and cell culture methods to recover viable organisms, or through newer, more rapid techniques using PCR to detect any mycoplasma DNA that is present within a sample,” Smith said.
The Eurofins scientists test cultures for mycoplasma with MycoSEQ from Thermo Fisher Scientific. This test can be completed “within a day and yield results within 24 hours of testing,” Smith explained. “However, this does not mean that the mycoplasma were viable at the time of testing, only that mycoplasma DNA is present within the sample.” Although PCR-based methods provide enough sensitivity, Smith noted that “a small percentage of cell-line types may interfere with the ability of the PCR method to detect mycoplasma DNA.” Consequently, he said, “Interference testing is therefore critical in conjunction with mycoplasma detection by PCR.”’
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Other vendors also make PCR-based kits for this bacteria. As an example, this video shows how to use the PromoCell Mycoplasma Test Kit. Jürgen Becker, product manager for cell biology at PromoCell, called this kit “convenient, highly sensitive, and reliable.” He added that these kits “detect all mycoplasma species relevant for cell culture contamination.”
Image: Jürgen Becker doing a mycoplasma test in PromoCell’s training academy.
Sartorius manufactures PCR-based test kits that can reveal the presence of mycoplasma in just three hours. “The earlier you detect mycoplasma contamination, the higher your long-term savings,” the Sartorius website notes. “Early detection means more time to react.”
Different kits can be used in different applications. As an example, Sartorius notes that its “Microsart AMP Mycoplasma is suitable for sensitive and robust detection of mycoplasma contamination in pharmaceutical processes.”
The traditional cell culture techniques take days to test for mMycoplasma, but they show if the bacteria is alive or not. Still, Smith said, “due to the short length of testing needed, PCR methods are the choice of mycoplasma detection testing in most applications, especially for autologous cell therapies.”
Keeping it under control
If any culture tests positive for mycoplasma, scientists need to decide how to react. “The best policy for cell lines contaminated with mycoplasma is to destroy the cell line and start with a fresh harvest of a cell line from a vetted source,” Smith recommended. “This is not always possible, due to rarity of a cell type or the amount of development that went into a cell line.”
When an infected cell line cannot just be discarded, scientists need another approach. Becker recommended treating a contaminated culture with “mycoplasma-effective antibiotics, such as PromoCell’s BIOMYCs, or using specifically designed kits that combine mycoplasma growth inhibition with highly efficient direct killing of mycoplasmas in cell culture, such as their Mycoplasma-EX Kit.”
If a cell line needs to be maintained, but had to be treated for mycoplasma infection, special follow-up steps must be taken. “It should be tested extensively through multiple passages after the decontamination has been attempted,” Smith said. “Only then can it be deemed to be cleared of mycoplasma contamination.”
From the start, though, scientists and companies should strive to prevent contamination in cell lines. Becker suggested “regular testing of cell cultures for mycoplasma contamination, as well as testing all cell cultures you are starting with and that have been frozen, in stock or have been donated to you by colleagues, other institutes etcetera.” He also encouraged scientists to follow “good laboratory practices, meaning to avoid conditions where infection of cell culture and spreading of contamination can occur.”
To avoid problems, scientists must use good disinfection procedures. As Becker recommended: “Use reliable, highly effective disinfectants, such as our surface disinfectants, Spore-EX and Mycoplasma-ExS, and our water disinfectants, Aquaguard, which are non-toxic, non-volatile, and non-corrosive and thus ideally suited for routine use.”
Smith also made a few suggestions for controlling infection. “The best controls are extensive testing of new cell lines, reagents, and raw materials used in the development and production processes, as well as good aseptic technique and environmental controls while handling cell lines for further processing,” he said. “The more critical a cell line is for production, the more often that cell line should be tested, during production, for mycoplasma contamination.”
Like any culturing process, preventing contamination from mycoplasma requires careful and consistent technique. In general, cultures must be regularly tested for bacterial infection. That ongoing effort can prevent disastrous problems down the culture road.