Mycoplasma contamination: minimizing risks to avoid cell culture complications


Microbial contamination of mammalian cell culture not only threatens the viability of the culture itself, it raises questions about the integrity of the process and the validity of the resulting data or products. The nature of the cell culture environment means that it coincidentally satisfies the nutritional and growth requirements of many different microorganisms, all of which can present a potential risk. As a result, strict aseptic techniques must be applied in conjunction with the implementation of a series of measures designed to help prevent or detect and rapidly eliminate any contamination.

Among the many potential contaminants, mycoplasma species present a particular challenge. This is mainly because their extremely small size makes them both difficult to exclude from the cell culture environment and difficult to detect. Understanding the risks involved helps guide mitigation strategies, which include rigorous asepsis, intensive filtration of reagents, and regular use of the most effective detection methods.

The problem of mycoplasmas

Mycoplasmas are the smallest known living organisms. They have long been recognized as common contaminants in mammalian cell cultures and remain a significant challenge in today’s laboratories and biopharmaceutical production facilities. In a 2015 study designed to obtain an unbiased assessment from multiple laboratories, analysis of sequence data from 9,395 rodent and primate samples from 884 series in the NCBI’s sequence reading archive, showed that 11% were contaminated with mycoplasmas.1

Mycoplasmas are distinguished from other bacteria by their extremely small physical size (0.15 to 0.3 µm), their small genome (580 to 2200 kb) and the absence of a cell wall. The combination of their size and pleomorphism allows these organisms to pass through standard sterilizing grade 0.2 µm filters. Since they can then reach high concentrations without creating noticeable turbidity, the effects of contamination are generally not visible to the naked eye until damage is done. The lack of a cell wall means that they are not sensitive to commonly used cell culture antibiotics, making their eradication difficult. To complicate matters further, antibiotics that are effective can be toxic to the cell culture itself.

Contamination with mycoplasmas can have several different origins. Laboratory personnel are considered a major source with more than half of all mycoplasma infections in cell cultures resulting from organisms typically found in the human oropharyngeal tract.2 Cell culture additives of animal origin, and in particular materials of plant origin, can all give rise to other types of mycoplasmas. Existing contaminated cell lines and equipment also pose a serious threat.

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Mycoplasma contamination can persist undetected for many years in cell cultures.3 Although it does not always cause cell death, it can alter the behavior of host cells. This can lead to changes in metabolism and cell morphology, chromosomal damage and aberrations, and an increasing tendency for apoptosis. There may be decreased rates of cell proliferation, reduced saturation density, and agglutination in suspension cultures.

As a result, it is not possible to rely on experimental data from contaminated cultures, making rework an absolute requirement. Once a laboratory has confirmed mycoplasma contamination, it will also need to perform a thorough disinfection of all laboratory materials, equipment and surfaces. Often, large quantities of material which may have been contaminated by contact must be discarded. Mycoplasma contamination of cell cultures therefore has enormous financial and productivity implications for laboratories and production facilities.

Preventive strategies

Managing the risk of mycoplasma contamination relies on maintaining the highest standards of aseptic technique and good hygienic practices. It is also recommended that one or more of the following measures be taken: filter all cell culture materials to less than 0.1 µm, rather than the more conventional 0.2 µm, which lacks mycoplasmas; purchase only 0.1 µm pre-filtered material; regular tests with techniques for detecting mycoplasmas which allow early action in the event of contamination.

Historically, filtration of cell culture media and components has been done using filters with a pore size of 0.2 µm, but there is now a slippage towards 0.1 µm. One of the main drivers of these stricter specifications is the increasing move towards plant-based cell culture materials, which are in fact more prone to mycoplasma contamination than those derived from, for example, albumin. bovine serum (BSA).4 Image 1 shows a typical vacuum filter configuration in which a modern design ensures high filtration efficiency, fast flow rates and high flow rate which are essential to maintain productivity in cell culture laboratories, while playing a key role in mitigating the risk of contamination.

Image 1: A typical filtration setup applicable to cell culture materials using 0.1 µm pore size. Credit: Thermo Fisher Scientific.

Today, a growing range of cell culture media and components are available as pre-filtered products. When purchasing them, it is important to ensure that all materials have been filtered to 0.1 µm. As would be expected, these products may charge higher prices. Facilities using less common cell culture media, custom media, or media with additional components cannot always achieve pre-filtered products and therefore must perform in-place filtration. Many people take a combination of approaches.

The importance of regular testing

Taking preventative measures to avoid mycoplasma contamination offers the best practices for managing cell cultures, and regular testing to verify the effectiveness of these measures is essential. The only sure way to detect mycoplasma contamination is to test cell cultures directly.

All tests are essentially reactive; once the mycoplasma contamination is identified, the culture is already ruined and any results should be discarded. This problem is exacerbated by the challenges of using traditional microbiological tests for mycoplasmas. These are long and the results can take up to 28 days. The latest rapid methods, however, allow for faster confirmation of contamination so researchers know immediately to discard a batch of cell culture, take the necessary corrective disinfection action, and start over.

Such damage limitation means less work that has to be repeated afterwards. PCR-based DNA testing is now an accepted method for detecting mycoplasmas in cell culture and provides complete results in just a few hours. Image 2 shows typical sampling points throughout a cell culture process. This early detection not only minimizes necessary rework, but also enables rapid action to protect against the spread of mycoplasmas in downstream equipment, processes and media.

Image 2: Sampling points for mycoplasmas. Rapid PCR-based tests for mycoplasma infection can be performed throughout the cell culture manufacturing process, from inoculation to harvest. Credit: Thermo Fisher Scientific.

In conclusion

Mycoplasma contamination is an insidious problem in mammalian cell culture. Mitigation of risk requires the adoption of strict aseptic practices and is supported by the application of rigorous media filtration criteria and the routine use of rapid PCR-based tests.

Without a doubt, prevention is better than repair. More efficient filtration of media and components down to a membrane pore size of 0.1 µm is becoming the norm, avoiding the problems associated with using standard 0.2 µm sterilization grade filters, which do not ‘do not prevent the passage of mycoplasmas. High-flow and high-flow filtration systems make it easier for laboratories to implement preventive strategies.

At the same time, there is the increasing commercial availability of pre-filtered materials. These validated and certified materials eliminate the need for additional filtration. For many, however, pre-filtered cell culture media may not be an option. Field filtration is still required for custom media and specialty materials, for example, or to meet local specifications.

Finally, the advent and acceptance of rapid tests for mycoplasmas means that laboratories can easily perform regular cell culture tests and know almost immediately if there is a contamination problem. They can then act quickly to eliminate affected crops and prevent further spread. Rapid testing – especially compared to traditional 28-day results with standard microbiological testing – minimizes loss of work and the need for rework.

The references:

  1. Olarerin-George AO, Hogenesch JB. Assessment of the prevalence of mycoplasma contamination in cell culture via a survey of the NCBI RNA-seq archives. Res nucleic acids. 2015; 43 (5): 2535-2542. do I: 10.1093 / nar / gkv136
  2. Nikfarjam L, Farzaneh P. Prevention and detection of mycoplasma contamination in cell culture. J cell. 2021; 13 (4): 203-212. PMCID: PMC3584481
  3. Uphoff CC, Drexler HG. Detection of mycoplasma contamination in cell cultures by polymerase chain reaction. In: Cree I. (eds) Cancer cell culture. Methods in molecular biology (methods and protocols). 2011. Humana Press. do I: 10.1007 / 978-1-61779-080-5_8
  4. Akers J, Meltzer T, Jornitz MW. The reappearance of mycoplasma contamination: prevention strategies. Bioprocesses International. Posted February 1, 2009. Accessed November 17, 2021


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