Mycoplasma is perhaps the most insidious type of bacteria faced by cell culturists. Mycoplasma are too small to see with most cell culture microscopes. They often don’t alter the cell lines in obvious ways, so contamination is not apparent. They lack a cell wall, rendering common antibiotics ineffective against them. They are small enough to evade the usual 0.2-micron filters used for cell culture preparations. They are all over human skin, so any breach in proper cell culture techniques can result in contamination.
Yet contamination is not uncommon. And worse—cell lines infected with mycoplasma can give erroneous results. Not only do mycoplasma compete for nutrients with cultured cells, but they also secrete toxins that cause additional stress. As a result, infection can lead to inhibition of many processes, including proliferation and protein synthesis. “People in the biopharmaceutical industry are concerned about this because it leads to much reduced productivity of their cell lines,” says Kai Nesemann, product manager at Sartorius. “And people in basic research are concerned because it makes them worry about reliability of their cell-based results.”
Cell lines infected with mycoplasma can give erroneous results.
There are several types of tests for mycoplasma contamination. The oldest test entails plating cell culture samples on agar and watching for growth of mycoplasma colonies. This test gives an unequivocal result, but also takes 28 days to complete. If you just received some eagerly awaited new cells, it’d be nice to use a faster test to check them for contamination—and then get on with your experiments without waiting a month. Here’s a look at some different types of mycoplasma tests that can help you decide what’s right for your lab.
Types of mycoplasma assays
PCR-based assays
These tests can use either conventional or real-time, quantitative PCR (qPCR). Both types detect the 16S ribosomal RNA of mycoplasma in several hours. Examples of conventional PCR-based assays include ATCC’s Universal Mycoplasma Detection Kit and Roche Custom Biotech’s MycoTOOL PCR Mycoplasma Detection Kit.
An example of a qPCR-based test is Thermo Fisher Scientific’s MycoSEQ™ Mycoplasma Detection Assay, which takes less than 5 hours and detects over 90 Mycoplasma, Acholeplasma, and Spiroplasma species. It was designed as a sensitive, specific, and rapid test to replace the traditional 28-day culture-based test for their primary users, who mainly come from the biopharma industry, cell and tissue therapy labs, and larger mammalian cell banking labs, says Mike Brewer, director of pharma analytics at Thermo Fisher Scientific.
Sartorius’s Microsart® line of mycoplasma testing kits also use qPCR, with highly specific TaqMan probes to detect even weak signals. “All users would like to have rapid and convenient testing, so we integrate the control reaction into the same reaction as the sample,” says Nesemann. “We developed a method where two fluorescent dyes are combined in one reaction, with one for mycoplasma contamination, and one for the internal control.” Another qPCR-based tool is Roche Custom Biotech’s MycoTOOL Mycoplasma Real-Time PCR Kit.
Biochemical assays
Biochemical tests, such as Lonza’s MycoAlert™ Mycoplasma Detection Kit, measure the activity of mycoplasma enzymes. MycoAlert uses a luciferase-based bioluminescence readout and takes less than 20 minutes. The MycoAlert PLUS Mycoplasma Detection Kit works the same way, but has increased light output that works with less sensitive plate readers.
Cell-based assays
InvivoGen’s PlasmoTest™ Mycoplasma Detection Kit also relies an alkaline phosphatase readout. It uses engineered HEK293 cells (HEK-Blue™-2 cells) as mycoplasma sensors that stably express a pathogen recognition receptor (TLR2). Upon binding mycoplasma, TLR2 initiates a signaling cascade that ultimately results in the secretion of alkaline phosphatase into the media. In the presence of HEK-Blue Detection medium, mycoplasma contamination is indicated by a media color change. This assay takes less than an hour of hands-on time, with an overnight incubation.
Nucleic acid tests
Some assays are based on the detection of nucleic acids with oligonucleotide probes, such as Bio-Techne’s MycoProbe Mycoplasma Detection Kit. This is a plate-based colorimetric assay that detects the mycoplasma 16S ribosomal RNA with PCR-level sensitivity in about 4.5 hours. “Since mycoplasma attaches to cells in culture, the best sensitivity is achieved from cell pellets that are lysed and tested for the absence or presence of ribosomal RNA,” says Birte Aggeler, director of antibody development at Bio-Techne. The microplate format enables high-throughput routine testing.
Prevention is the best medicine
Although it would be ideal to work only with cell lines certified as mycoplasma-free, that’s not always an option. Instead, says Aggeler, “I recommend a quarantine lab with a separate hood and incubator for new cell lines, until they have tested negative for contamination and can be brought to the general cell culture facility.” As for minimizing cross-contamination risk between scientists, Aggeler suggests not sharing media, and using filtered pipette tips. “For shared facilities, it’s crucial to clean cell culture hoods and equipment on a regular basis,” she says.
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Nesemann agrees that the best option is to prevent contamination in the first place. Regular testing, to detect any small infections before they spread to other cultures, is crucial. Attention to sterile technique is key, including filtering all media and raw materials with 0.1-micron filters, and adhering to strict user hygiene. “Use mycoplasma decontamination spray or wipes to decontaminate the glass, the bench, everything,” says Nesemann. “And many people don’t know this, but you also have to sterilize your liquid nitrogen devices, which, once infected, can quickly spread contamination.”
Toss it, or treat it?
In the event of contamination, the most conservative approach is to discard the infected cultures. But if a rare or valuable cell line becomes infected, it might be possible to rescue it by treating the infected cultures with an antimycocidal agent, such as Lonza’s MycoZap™ Mycoplasma Elimination Reagent. Unfortunately, this approach doesn’t always work. “Sometimes the mycoplasma can come back after a few cell generations post treatment,” says Brewer. “There’s a theory that a small number of mycoplasma may be harbored inside the mammalian cells, allowing them to survive the antimycocidal treatment.” If attempts are made to cure an infected cell line, it’s wise to follow up with rigorous mycoplasma testing for multiple generations after treatment. Even then, the infection or treatment could cause an alteration to the cell line that is difficult to detect.
Regular testing for success
When multiple researchers work with multiple cell lines, it’s fairly easy for contamination in one infected cell line to spread quickly to other cell lines through shared equipment, media, or even aerosols in the work area. Even though people can potentially introduce human-borne mycoplasma into cultures, Brewer says that “it’s not so much operator-introduced contamination, as unknowingly working with a highly contaminated cell line” that can spread mycoplasma through a lab. Without regular testing, the contamination can easily go unnoticed because it doesn’t cause any observable change to the cell culture. “So if you aren’t testing routinely, you can’t detect contamination and prevent its spread to other cultures,” says Brewer. He recommends testing any new cell line as soon as it is received, and then establishing a routine testing schedule. Frequency of testing will depend on the number of cell lines, users and other risk assessment factors.
If today’s newer and faster mycoplasma testing kits are too much work, you can also consider outsourcing this vital but admittedly tedious task. Many vendors or services offer routine mycoplasma testing services. One way or another, make sure the testing gets done!
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