Editorial Article
Monday December 14, 2009
by Jeffrey M. Perkel
Don't look now—actually, do!—but something may be lurking in your cell culture. Microscopically invisible, impervious to most common antibiotics, and causing no obvious changes to your culture media, a bacterial contaminant may be covertly wreaking havoc with your data.
I'm speaking, of course, of mycoplasma. Members of the family Mollicutes, mycoplasma comprise some 180 distinct species, six of which (Mycoplasma orale, M. arginini, M. fermentans, M. salivarum, M. hyorhinis, and Acholeplasma laidlawii) account for nearly 95% of all lab contaminations, according to Andrea Toell, senior product manager at Lonza, which offers a range of mycoplasma management products.
According to one study, incidence of mycoplasma contamination in cell culture labs ranges from 15% to 35%1; another study places the rate as high as 80% 2. Brian Douglass, product manager for cell biology at the American Type Culture Collection (ATCC) estimates that in his experience, for newly acquired strains grown outside ATCC, contamination is "common," affecting about 10% of cultures.
But even if the prevalence is closer to the low end of the range, it is still too high. Though infection won't typically kill cultured cells, it can subtly change their growth characteristics and biology—anything from metabolism and gene expression to chromosomal integrity and apoptotic potential, says Toell. "So, not knowing if you have a mycoplasma contamination, you can never be sure if the results you get from any assay arise from normal cell characteristics or from changes induced by mycoplasma," she explains.
Mycoplasma are among the smallest of Bacteria. They are free-living—that is, they grow outside of cells—yet unlike most bacteria and fungi, produce no visible changes in culture media (e.g., turbidity or pH changes). Being so small, mycoplasma cannot be seen under a microscope. Having no cell wall, they are immune to many common antibiotics. As a result, they can remain undetected and uninhibited for extended periods of time.
"[Mycoplasma] contaminations are a thorn in the side of everyone doing cell culture, as they often go undetected," says Don Finley, market segment manager for research cell culture at Sigma-Aldrich.
Fortunately, researchers have at their disposal a range of products for the prevention, detection, and treatment of mycoplasma contamination. All that is required is the discipline to use them regularly.
At the ATCC, says Douglass, mycoplasma is serious business. Dealing as it does with thousands of different cell lines, ATCC's approach could best be described as "old school," he says. The facility's six-member biology quality control team tests for contamination whenever it makes a "token," "seed," or "distribution" lot of any strain, Douglass says. Those QC officers have their work cut out for them: the ATCC models its Mycoplasma QC program on the US Food and Drug Administration's rigorous guidelines covering mycoplasma testing for viral vaccine development.3
Those guidelines specify two testing procedures, the "Agar and Broth Media Procedure" and the "Indicator Cell Culture Procedure," and ATCC does both, in duplicate. In the former assay, broth cultures are inoculated with culture supernatant to promote bacterial growth. On days 0, 3, 7, and 14, aliquots are removed and plated onto agar plates. These plates are then grown for an additional 14 days, after which they are examined for growth of mycoplasma. In the second procedure, Vero cells are inoculated with culture material and grown for three to five days, after which they are stained with Hoechst DNA stain and examined microscopically for cell surface-associated fluorescence.
Not every strain shows up in both tests. According to Douglass, the Agar and Broth Media Procedure catches most slow-growing mycoplasma; the Indicator Cell Culture Procedure tends to pick up faster-growing strains. Perhaps most notably, M. hyorhinis grows poorly in the agar assay, and can only be detected via Hoescht staining. "By combining these two tests, you're getting the most comprehensive picture of any potential mycoplasma contamination in your culture," he says.
The cost for that comprehensive picture, though, is time. The Agar and Broth assay may be the "gold standard" for mycoplasma detection, but at ATCC, which has staff and equipment dedicated to running these assays, it takes "five to six weeks" from inoculation to final results. The fluorescent assay is faster, but still requires several days, not to mention the growth of an additional dedicated cell line.
Thus, Douglass says, researchers who merely want to assess the state of their cultures, and who have neither the personnel, space, nor time for the more traditional methods, would probably do well to use some of the newer kits on the market—though he adds, for ambiguous results, "there's no better method than the broth and agar method."
"For throughput, cost, and space reasons, the kits on the market make perfect sense," he says.
Researchers have three options when it comes to kits, the most popular of which is probably PCR, with detection kits available from Roche Applied Science, Sigma-Aldrich, Life Technologies, and Agilent Technologies.
Sigma-Aldrich offers two PCR-based tests, says Finley, both of which use primers designed to target between 19 and 24 different Mycoplasma species based on 16S rRNA: the LookOut® Mycoplasma PCR Detection Kit and the VenorGeM® Mycoplasma Detection Kit, PCR-based. The first, he says, is part of the company's high-throughput, automation-ready "Cadillac" solution to mycoplasma control; the second is more of a manual, "VW" option.
Agilent offers three PCR-based tests: the Mycoplasma Plus™ PCR Primer Set, the MycoSensor™ PCR Assay Kit, and the MycoSensor QPCR Assay Kit. Like Sigma-Aldrich's detection kits, the MycoSensor assays also target 16S rRNA genes, says Mimi Ly, product manager for mutagenesis, cloning and protein expression at Agilent Technologies, Stratagene Products Division, while the Mycoplasma Plus PCR Primer Set detects a different (undisclosed), single-copy mycoplasma gene. But all three can detect at least eight Mycoplasma species, she says, which account for at least 95% of all infections.
The Mycoplasma Plus assay is a gel-based endpoint PCR assay that also uses Sau3A restriction digestion to not only verify the specificity of the amplification reaction and corroborate the results, but also identify which of five mycoplasma species is actually present. In contrast, the MycoSensor assays, based on SYBR® Green detection, are faster, and feature improved primer designs to prevent detection of E. coli DNA as false positives.
According to Ly, sales figures at Agilent suggest that endpoint PCR-based assays like the Mycoplasma Plus PCR assay are on the wane, whereas qPCR assays are ascendant—an observation she says makes sense given the growing acceptance of real-time PCR assays. "qPCR has many advantages," she notes; in particular, it is both quantitative and more sensitive than endpoint PCR. As a result, you can detect infections both earlier and at lower copy number.
The Applied Biosystems Myco Scan kit from Life Technologies also employs SYBR Green-based qPCR detection of mycoplasma DNA. A research-scale version of the company's biopharma testing kit, Myco Scan features "very high sensitivity, very broad species coverage, and optimized sample preparation," says product manager Surekha Karudapuram.
In fact, MycoScan detects more than 90 different species via a 31-primer multiplexed reaction. "We really spent a lot of bioinformatics and testing time to optimize this," Karudapuram says.
For those with more industrial needs, Roche's endpoint-based MycoTOOL "is the only methodology that involves PCR technology for Mycoplasma detection that has been approved for lot-release testing [by US, European, and Japanese regulatory agencies]," says Doug Irving, marketing manager for industrial business at Roche Applied Science. Indeed, at least nine FDA-approved biopharmaceutical products, including seven from Genentech, have been greenlit for release based partly on this test, Irving says.
Other options include Roche's Mycoplasma Detection Kit, based on ELISA, and Lonza's MycoAlert® Mycoplasma Detection Kit, which is an enzyme-based assay. According to Toell, MycoAlert uses the substrate for two mycoplasma-specific enzymes, which results in the production of ATP. That ATP, in turn, is detected by its ability to drive a luciferase reaction.
"It’s very quick, just 20 minutes to assay, and the enzymes we are detecting are found in all six of the main Mycoplasma cell culture contaminants and in most of the 180 species that have been determined," she says.
But the primary advantage of the MycoAlert test, she says, is that—unlike PCR-based methods—it can distinguish between dead and living cells, and also does not need to rely on universal PCR primer sets.
If you do detect contamination in your culture, the safest bet is to discard the contaminated cultures, thoroughly clean your incubators and hoods, and start fresh. But, if you are working with something precious, products are available to help rid a culture of infecting Mycoplasma, such as Lonza's MycoZap™ Elimination Reagent and Sigma-Aldrich's LookOut® Mycoplasma Elimination Kit. Once you've restored your cultures' integrity, other products facilitate routine mycoplasma prevention (e.g., Lonza's upcoming MycoZap Spray and MycoZap antibiotic formulations).
So, how often should you test? Every lab is different, says Agilent's Ly. Some do it monthly, others quarterly. Still others test whenever new lines come in—probably the most common route of infection (the others being contaminated sera and lab personnel). According to the recommendations of the Second ECVAM Task Force on Good Cell Culture Practice, "it is recommended that, as a minimum and where advisable, mycoplasma testing should be carried out on all samples received," the authors suggest. 4
References:
1 HG Drexler, CC Uphof, "Mycoplasma contamination of cell cultures: Incidence, sources, effects, detection, elimination, prevention," Cytotechnology 39: 75–90, 2002.
2Koshimizu K, Kotani H. In: Procedures for the Isolation and Identification of Human, Animal and Plant Mycoplasmas (Nakamura, M., ed.), Saikon, Tokyo, 87-102, 1981.
3 US FDA, "Draft Guidance for Industry: Characterization and Qualification of Cell Substrates and Other Biological Starting Materials Used in the Production of Viral Vaccines for the Prevention and Treatment of Infectious Diseases," 2006.
4S Coecke et al., "Guidance on Good Cell Culture Practice. A report of the Second ECVAM Task Force on Good Cell Culture Practice," ATLA 33:261-87, 2005.