Jeffrey Perkel has been a scientific writer and editor since 2000. He holds a PhD in Cell and Molecular Biology from the University of Pennsylvania, and did postdoctoral work at the University of Pennsylvania and at Harvard Medical School.
Researchers in Barbara Ehrlich’s lab at Yale University, in collaboration with David Kaplan’s lab at Tufts University, were about eight months into a study of 3D kidney cyst formation when they made an unpleasant discovery. The porcine kidney cells upon which the project was based were riddled with bacteria. Specifically, mycoplasma—a family of tiny intracellular pathogens that are immune to most common antibiotics, invisible to the naked eye and can wreak havoc on cultured eukaryotic cells.
“We were, to put it frankly, freaked out,” says Ivana Kuo, an associate research scientist in the Ehrlich lab, who was heading up the study alongside Teresa DesRochers, a senior scientist in the Kaplan lab at the time.
Upon reflection, the researchers decided they had no choice but to redo their initial work—a process that ultimately took about a year from decontamination to publication [1]. But, in what could be the ultimate demonstration of turning lemons into scientific lemonade, they also studied the contaminated cells, measuring the impact of mycoplasma contamination on 3D cell growth [2].
Their conclusion, published separately in PLOS ONE: That though mycoplasma infection seemed to have no measurable impact upon their cells when grown in 2D cultures, it did affect the number, size and integrity of cysts that formed in 3D. Among other things, Kuo says, the cell-cell junctions seemed “leakier,” which presumably would negatively affect the cells’ ability to communicate.
Kuo’s experience, unfortunately, is hardly unique. Some 15% to 35% of cell cultures are contaminated with mycoplasma, according to one 2002 estimate [3]. For cell biologists, a mycoplasma contamination not only impacts the quality of the research but also is costly—both financially and in terms of scientists’ precious time. Fortunately, there exists a robust set of tools to detect, eliminate and prevent infection.
Screening methods
According to Bob Geraghty, biorepository and cell services head at the Cancer Research UK Cambridge Institute, who coauthored a detailed guide to cell culture in 2014 [4], the “gold standard” assay for mycoplasma detection is direct culture on agar plates. Mycoplasma yields characteristic “fried egg”-shaped small colonies [3]. But it can take weeks for those colonies to grow, requiring the long-term cultivation of unwelcome contaminants. As a result, Geraghty recommends that researchers interested in this particular assay outsource the work to specialized testing labs.
PCR-based assays—which test for characteristic 16S ribosomal RNA or other genetic signatures—represent one popular alternative. Cord Uphoff, a staff scientist at the Leibniz Institute DSMZ- German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany, uses PCR in his lab. “It’s the most sensitive and also a specific test,” he says. But controls are key, he stresses, as both false positives and negatives can arise, from cross-contamination or polymerase inhibitors in the growth medium, respectively.
Other options include assays for mycoplasma enzyme activity (e.g., Lonza’s MycoAlert™ bioluminescence assay), assays based on microscopic detection of mycoplasma DNA in cells (e.g., via in situ hybridization or DAPI staining) and ELISA-style nucleic acid detection assays, including R&D Systems’ MycoProbe® Mycoplasma Detection Kit and Roche’s Mycoplasma PCR ELISA (distributed by MilliporeSigma). Geraghty’s lab uses the MycoProbe assay, which detects mycoplasma RNA, for routine screening and follows up with PCR to double-check any positive results.
Those using nucleic acid-based assays can calibrate their processes with the World Health Organization (WHO) “international standard for mycoplasma DNA,” says Micha Nübling, head of the WHO Blood Group, which developed the standard [5]. Though developed specifically for testing labs, individual researchers also can use this reagent—available from the Paul-Ehrlich-Institut—as “a reference preparation,” Nübling says, for instance to determine detection limits.
In the event contamination is discovered, the safe choice is simply to discard the culture and start fresh.
“The first question is, is this [culture] absolutely irreplaceable?” says Amanda Capes-Davis, founding manager and honorary scientist at CellBank Australia. “If it isn’t, throw it away and get it from somewhere else. Because if it has mycoplasma, it may have other problems, as well.”
Rescuing your cells
If the culture is irreplaceable, it can be cleaned of mycoplasma using any of a variety of commercial formulations [6]. Uphoff, for instance, recommends using three different treatments in parallel—a fluoroquinolone, such as Ciprobay; a mixture of pleuromutilin and tetracycline (BM-Cyclin); and a mixture of a macrolide and another fluoroquinolone (Plasmocin). As many as a quarter of known mycoplasma are resistant to at least some of these treatments, Uphoff explains. But by using them in parallel, it’s a good bet that at least one approach will succeed.
Though instructions vary for each reagent, the cleaning process generally involves treating the cultures for several passages, followed by a recovery period without treatment, at which point the cells should be tested for residual contamination. Lonza’s MycoZap reagent, for instance, requires four cell passages, according to senior product manager Andrea Toell. Thus, cells that are passaged every two to five days will take eight to 20 days to clean.
After cleaning, be sure to test the cells thoroughly, says Geraghty—anti-mycoplasma treatments can be cytotoxic, so the cells may well behave differently. Amy Noble, product manager for mycoplasma detection and elimination reagents at MilliporeSigma, recommends karyotyping the cells to ensure no gross chromosomal abnormalities have arisen.
Of course, when it comes to mycoplasma, the best offense is a good defense.
Obtain cell lines from trusted vendors that test for mycoplasma, such as the ATCC (American Type Culture collection) or ECACC (European Collection of Authenticated Cell Cultures). If you need to obtain cells from other labs, quarantine and test them immediately—Geraghty advises requesting two frozen vials and testing one directly, without culturing it—and then grow and freeze down stocks that can serve as a clean, internal cell stock. “Approach this with a cell-banking approach,” advises Capes-Davis.
Most importantly, all agree, focus on your aseptic technique. The vast majority of mycoplasma contamination results from cross-contamination from infected cultures, or from human skin. Thus, good culturing techniques—using gloves and a clean lab coat and wiping down the hood between different cultures—are a must.
It is important to get into the practice of testing your cultures frequently, at least quarterly if not monthly, and to clean your hoods and incubators routinely. Prior to its brush with mycoplasma, the Ehrlich lab tested its cells annually, Kuo says; they’ve since changed that to three times a year and have yet to see a recurrence. Says Geraghty, “What you don’t want to do is get to the end of some lengthy bit of research, only to find out your cells are positive.”
References
[1] Kuo, IY, et al., “Cyst formation following disruption of intracellular calcium signaling,” Proc Natl Acad Sci, 111:14283-8, 2014. [PMID: 25228769]
[2] DesRochers, TM, et al., “The effects of mycoplasma contamination upon the ability to form bioengineered 3D kidney cysts,” PLOS ONE, 10:e0120097, 2015. [PMID: 25793639]
[3] Drexler, HG, Uphoff, CC, “Mycoplasma contamination of cell cultures: Incidence, sources, effects, detection, elimination, prevention,” Cytotechnology, 39:75-90, 2002. [PMID: 19003295]
[4] Geraghty, RJ, et al., “Guidelines for the use of cell lines in biomedical research,” Br J Cancer, 111:1021-46, 2014. [PMID: 25117809]
[5] Nübling, CM, “World Health Organization international standard to harmonize assays for detection of mycoplasma DNA,” Appl Env Microbiol, 81:5694-5702, 2015. [PMID: 26070671]
[6] Uphoff, CC, Drexler, HG, “Eradication of mycoplasma contaminations from cell cultures,” Curr Protocols Mol Biol, 28.5.1–28.5.12, April 2014. [PMID: 24733241]