Cell-based assays have become a vital tool for drug discovery and an emerging trend in other markets as they allow scientists to study true cellular responses and behaviors under conditions that mimic the cell’s natural environment. Much attention has been focused on further miniaturization and increased sensitivity of these assays; however, one critical and basic element of cell-based assays must not be overlooked or underestimated — the dangers of mycoplasma contamination.

Treatment for mycoplasma contamination is laborious, costly, and at times ineffective; in fact, many choose to dispose of the cells completely and start with a new culture. In order to effectively prevent and contend with this threat, it’s important to understand what mycoplasma is and how it affects cell cultures.

Defining mycoplasma

Mycoplasma are classified as prokaryotes with several unique properties that distinguish them from other prokaryotes. They lack a cell wall, instead using sterols, especially cholesterol, from vertebrate hosts to maintain their plasma membrane. Therefore, they are unaffected by antibiotics that interfere with the murein formation of cell walls. It also means that mycoplasma do not overgrow their host cells but in fact bind with cell walls to obtain nutrients. As they are extremely small (0.15–0.3 μm), these organisms are difficult to filter out of suspension, and can grow to particularly high concentrations in mammalian cell cultures without producing turbidity or other obvious symptoms.

Mycoplasma grow very slowly as they infect the cell culture. Nonetheless, this can have a serious effect on the host cell’s metabolism. Unexpected alterations can develop in growth, metabolism, function, secretion and synthesis, and expression of the host cell. Additionally, damage can occur to the host cell’s membranes, DNA and RNA, and other intracellular organelles. All of this can lead to skewed and/or inaccurate data and, if not recognized as a mycoplasma infection, can seriously compromise the validity of the final product or study results.

Methods of transmission

The most widespread methods of mycoplasma contamination are cross-contamination of healthy cells with infected cultures and poor aseptic technique.

Cross-contamination can occur when untested, infected cells or media materials come into contact with a clean cell culture. Previously infected cells can come from other research laboratories as shared research, donations or gifts, or from commercial suppliers. Cross-contamination can also occur in conjunction with poor aseptic technique via aerosolization during routine handling of cells. Even in the protective environment of a laminar flow hood, airborne particles and aerosols can result from pipetting, dust in protective garments, dry, flaking skin, or even talking or sneezing while working around cell cultures. Mycoplasma contamination can often be recovered from equipment and media bottles used in a hood and even the internal surface of the hood several weeks after exposure to the contaminants.

Another example of poor aseptic technique that can result in mycoplasma contamination is the handling of supplies and equipment. Improperly sterilized or stored consumable supplies, media, or other materials may become contaminated before or during use. Materials and equipment stored in laminar flow hoods can disrupt air flow patterns, and increase surface area for potential mycoplasma contamination. Incubators with integrated fans and air currents created during opening and closing of the internal incubator door may spread mycoplasma-containing particles. Water baths, waste containers, and even cooling coils on refrigerators and freezers are major and often overlooked sources of mycoplasma contamination.

While sera and other animal-derived products, historically a common source of mycoplasma contamination, have improved dramatically in quality, they cannot be automatically eliminated as a source of contamination, especially if they are from an unknown or questionable manufacturing source.

Prevention

The single most important factor in prevention of contamination is proper aseptic cell culture technique. This reduces the risk not only of mycoplasma infection, but bacterial, viral, chemical, and other contaminants as well. The following guidelines can help to reduce the risk of contamination.

Reduce aerosol generation and airflow patterns

Wear dedicated personal protective equipment to shield cells from aerosol and debris contamination caused by street clothes, skin, hair, and even breathing. Clear work surfaces and laminar flow hoods of all clutter and storage boxes, and thoroughly clean with a suitable disinfectant between uses, and only use these surfaces and hoods for one cell line at a time. Laminar flow hoods should continually run unless they won’t be used for extended periods of time. To reduce turbulent airflow in the laboratory, the number of people in the laboratory should be as few as possible.

Increase attention to detail

Maintain and separately store media and reagents for each cell line and clearly label to eliminate any doubt or confusion. Clearly label the cell lines as well and test prior to and after freezing in a cryogenic cell repository to ensure that they are free from mycoplasma contamination. Additionally, rotate the frozen stock periodically to reduce dependence on the active cell culture. Clean, service, and calibrate incubators, water baths, environmental monitoring tools, and other equipment at regular intervals. Similar attention to care and cleaning should apply to the surrounding environment, including areas behind and underneath equipment, storage shelves and cabinets, and even low-traffic sections of the laboratory.

Perform routine testing

Test, test, and test again! Cell cultures should be tested for mycoplasma contamination on a regular basis, depending on the needs of each laboratory. Media and reagents should also be subject to rigorous testing prior to use. Any new cells, media, or reagents should be quarantined until they have tested free from mycoplasma contamination. Even equipment and work surfaces should be monitored and tested for exposure to mycoplasma.

Consider the human factor

Unwavering perfection is just not practical; accidents due to human nature are often unavoidable. Mistakes are much more likely to happen when operators are inexperienced, feel stressed, overworked, distracted, or rushed. Proper and repeated training sessions educate personnel on correct aseptic technique in cell culture, and also keep the lessons and skills learned top of mind. Additionally, being mindful of and focusing on the tasks at hand can prevent countless errors.

Use of antibiotics

Subject to occasional controversy, antibiotic use in cell culture can provide a benefit to preventing or eliminating bacterial contamination. However, when overused, antibiotics can provide a false sense of security and mask mycoplasma contamination. It can also promote resistance, even to antibiotics specifically targeted for mycoplasma infections. Antibiotics should be used with great care and only when absolutely necessary.

Mycoplasma detection methods

There are a number of mycoplasma detection methods that fall into two broad categories: direct testing and indirect testing for specific mycoplasma characteristics. Direct culture testing is the most sensitive method of cultivatable mycoplasma detection but also the most cumbersome and time-consuming; results can take up to four weeks. As it requires live mycoplasma controls, many laboratories prefer to reduce risk by using an outside testing facility.

Indirect methods, including biochemical and fluorescent assays, nucleic acid hybridization, immunoassays, and polymerase chain reaction (PCR) can detect both cultivatable and noncultivatable mycoplasma strains in much less time than direct methods. Biochemical and fluorescent assays are quick and userfriendly tests that measure mycoplasma enzyme activity. Results can be easily analyzed and documented via a multi-mode microplate reader in 30 minutes or less. Nucleic acid hybridization uses a chemiluminescent label to detect ribosomal RNA from mycoplasma; tests take over an hour to complete. Fluorescent assays require a microscope with particular UV filters to detect mycoplasma DNA, results are provided within 24 hours. Immunoassays measure specific mycoplasma antibodies, and deliver responses within hours, but must be type-specific. Finally, PCR protocols using primers to amplify ribosomal RNA from mycoplasma are highly sensitive and rapid, with results in about two hours, but involve complex handling protocols and may produce false positives.

The combination of a direct culture with an indirect test is recommended to ensure accurate results.

Remediation for contaminated cultures

If a culture is inadvertently contaminated with mycoplasma, two solution options are available. The first option, quick and easy, is simply to autoclave the culture, dispose of it, and start with a new culture. The second, riskier option is to treat the culture with specific antibiotics or other chemicals that are toxic to mycoplasma but safe for cells.

Proper antibiotic treatment should kill mycoplasma rather than inhibit growth. Unfortunately, complications can arise due to antibiotic resistance, cellular toxicity, or cytotoxic side effects to the cell culture itself. Most typical cell culture antibiotics are not effective against mycoplasma contamination but other types have shown success in eliminating mycoplasma from cell cultures. Ciprofloxacin, BM-Cyclin and quinolone derivatives provide reasonable mycoplasma elimination rates but none are proven to be 100% effective.

Non-antibiotic treatments can target mycoplasma by damaging the plasma membrane. However, they may also have cytotoxic effects to the cell culture, and to be effective must come in direct contact with the mycoplasma organism; reduced effects may be seen in adherent and clustered cells.

Summary

Cell-based assays are increasingly valuable as they offer more functional information than any other methods to date. As researchers strive to produce more effective results in less time, focus must not be diverted away from the hazards of mycoplasma contamination. As the most significant cell culture contaminant in the world, prevention, early detection, and successful eradication of this hazard is crucial to accurate and streamlined research.

Paul Held, Ph.D. is a Senior Scientist at BioTek Instruments, Inc., Highland Park, P.O. Box 998, Winooski, VT 05404-5171; (888) 451-5171; Heldp@biotek.com; www.biotek.com.