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  4. Mycoplasma Contamination in Cell Culture

What are mycoplasma

Mycoplasma species are widespread in nature as parasites of mammals (including humans), reptiles, insects, and plants. They are the smallest and simplest self-replicating prokaryotes at just 0.2–0.8 µm in size. Therefore, you can’t detect them under the light microscope and they also can pass through sterile filters. With a genome of only 0.58–2.20 Mb, Mycoplasma species have limited biosynthetic capabilities and are therefore dependent on their hosts for most nutrients.

Mycoplasma species belong to the Mollicutes class, which includes Acholeplasma and Ureaplasma amongst others. Mollicutes are gram-positive bacteria but unlike other species, they lack a cell wall and thus can adopt various different shapes. For convenience, the term mycoplasma is often used to refer to all species of the Mollicutes class.

Where does mycoplasma contamination come from?

There are six species that account for 95% of all detected mycoplasma contaminations in continuous cell line cultures: M. orale, M. arginini, M. fermentans, M. hyorhinis, M. hominis, and A. laidlawii. Typical routes of contamination are: cross-contamination from other cells (for example, via aerosols generated during pipetting), using the same media bottles, or handling more than one cell type at a time.

Other sources of contamination within your cell cultures could include direct contamination from the researcher, as well as the use of contaminated materials, such as animal sera. The high risk of contamination highlights the importance of always purchasing high-quality cell culture media from reputable manufacturers.

Our infographic provides you with 10 tips that help to keep your cells free from mycoplasma.

Top 10 tips for Mycoplasma
free cell culture

Infographic

Learn how to prevent your cell culture from Mycoplasma contamination.

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How can mycoplasma contamination affect your cell cultures?

Contamination of cell cultures with mycoplasma species can affect your cells in numerous ways, most of which will reduce the reliability, reproducibility, and consistency of your experimental results.

The type and severity of these effects will often depend on the cell type you are using. Learn more about mycoplasma contamination in cell culture by watching our webinar “Know your enemy – Mycoplasma contamination in cell culture”.
 

Adverse effects of mycoplasma on cell cultures include:

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  • Alterations to the levels of protein, RNA, and DNA synthesis due to nutrient deprivation
  • Chromosomal aberration
  • Alteration of DNA transfection efficiency
  • DNA fragmentation due to mycoplasmal nucleases
  • Disruption of nucleic acid synthesis
  • Changes in cellular morphology
  • Stunted, abnormal or inhibition of growth (3)
  • An increase (or decrease) in cytokine expression
  • Changes in cell membrane antigenicity
  • Inhibition of cell metabolism
  • Cell death

How can mycoplasma
contamination affect manufacturing
of biological products?

Contamination with certain mycoplasma species can also cause cytopathic effects, characterized by stunted, abnormal growth and degenerated cells, likely due to the promotion or inhibition of apoptosis.

This is a major problem for research, as well as during the manufacturing and processing of biological products and biopharmaceuticals because contamination can result in reduced yields or loss of an entire product batch. In addition, hybridoma cells, typically generated to support applications like antibody manufacturing, can show characteristic effects when contaminated with mycoplasma, such as inhibition of cell fusion and a lower yield of monoclonal antibodies.

Know Your Enemy – Mycoplasma Contamination in Cell Culture

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How can you detect mycoplasma contamination in your cell cultures?

Unfortunately, it is challenging to quickly and reliably detect mycoplasma contamination in cell cultures. One reason is that mycoplasma contamination does not typically trigger the turbidity changes that are common with other types of bacterial or fungal contamination. In addition, the bacteria are too small to observe using optical microscopy without the need for specific labeling.

However, cell cultures can show subtle changes as a result of mycoplasma contamination, meaning careful analysis can provide some insight. For example, as the mycoplasma will compete with your cells for nutrients in the culture medium, one of the first visible signs of contamination is a reduced rate of cell proliferation. Other indications of contamination include cell aggregation, morphological changes, and poor transfection efficiencies (which can be easier to spot when you are working with cells that used to show high transfection efficiencies in the past).

The only way to detect mycoplasma species is to explicitly test for them. There are several different techniques to identify if your cell cultures are contaminated with mycoplasma. These include histochemical staining, ELISA, DNA fluorochrome staining, microbiological culture, biochemical / bioluminescence methods, and PCR. A research group even managed to show the presence of Mycoplasma species by light microscopy using oblique illumination, which renders the mycoplasma cells visible as white foci due to light scattering.

The best mycoplasma assays are highly sensitive and specific, but can also be performed quickly. Based on these criteria, we discuss the most commonly used assays below.

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Your cell culture is
contaminated with mycoplasma,
what can I you do?

All cell culture contaminants can be problematic, but in comparison to other types of bacterial and fungal contaminants, mycoplasma contamination is usually more difficult to detect. This is because Mycoplasma are very small and replicate at a slower rate compared to many other bacterial species.

The presence of Mycoplasma in your cell cultures can seriously affect the behavior of your cells, making your results unreliable and unreproducible. We understand how frustrating it is to discover that your cultures are contaminated, especially as you might have to set up another culture and/or repeat any experiments performed since the last negative mycoplasma test. However, if you wish to obtain accurate data that is as representative as possible of your model system, it is usually best to discard the contaminated cultures and set up new ones.

You can continue with your experiments from the last negative mycoplasma test. Review guidelines for sterile working, including keeping your equipment in the best condition to reduce the risks of mycoplasma contamination, for example, in your incubators and water baths. This will identify where you can improve your facilities and procedures, helping you to reduce the risks of future mycoplasma contamination.

Explore our MycoAlert® Mycoplasma Detection Kits

Lonza’s mycoplasma detection kits are intended for the quick and convenient detection of mycoplasma in cell cultures.

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What options do you have when your cell cultures are contaminated with mycoplasma?

Elimination of mycoplasma contamination can be carried out by using specially formulated reagents, such as MycoZap® Mycoplasma Elimination Reagent. This tool combines antibiotic and antimetabolic agents to eliminate detectable mycoplasma contamination in as little as four days. It has been optimized to clear the contamination with minimal effects to your cell culture. More details on the elimination of Mycoplasma species can be found in the next section.

There are various other methods for eliminating mycoplasma contamination. These fall into one of four categories: chemotherapeutic, chemical, immunological, and physical. Elimination techniques include antibiotic treatment, exposure to detergents, exposure to complement, cell cloning, heat treatment, and filtration. Unfortunately, many of these techniques have been shown to be unreliable, as they cannot kill all Mycoplasma species. They are also very time-consuming and are not very efficient at removing contamination. Antibiotic treatment without antimetabolic agents, for instance, has shown promising results in mycoplasma-contaminated leukemia-lymphoma cell lines. However, in 3 – 20% of cultures, the mycoplasma bacteria were resistant, and in 3 – 11% of treatments, cytotoxicity was observed.

If antibiotics alone are unable to clear the mycoplasma contamination, bacteriostatic antimicrobial agents might help to inhibit the growth of the bacteria. Another approach would be to first try one antibiotic, and if this isn’t effective, use another antibiotic on a backup positive culture (i.e. one that you had stored separately, before your first attempt at eliminating the contamination).

How can you prevent mycoplasma contamination?

Although you will never have a 100% guarantee that your experiments are mycoplasma free, you can implement good working practices to limit the chances of contamination, while also reducing the risk of spreading contamination to other cultures. Mycoplasma contamination, transmission, and prevention require a three-pronged approach to ensure you are working as cleanly as possible1:

  1. Use the correct cell culture reagents and equipment

    It is essential that your facility offers suitable equipment, and that the laboratory is kept clean and tidy. This includes having a certified laminar-flow biological safety cabinet, performing regular inspections and cleaning of incubators, and ensuring the proper disposal of consumables and cell culture materials.

  2. Review and optimize your cell culture procedures

    Use reliable mycoplasma detection methods and test cultures on a regular basis to help limit the impact that contaminants have on your facility, as well as on your work. Keep incoming cultures in quarantine until proven to be mycoplasma free, and immediately discard contaminated cultures (if possible). Purchase cultures from reputable suppliers. Although overuse of antibiotics is not recommended, for precious cell cultures the continuous use of antibiotics that are effective against Mycoplasma species, such as MycoZap® Antibiotics, can be a way of preventing contamination.

  3. Ensure you adopt aseptic techniquess

    Thorough washing and disinfecting of your hands is key to working as cleanly as possible. Wear personal protective equipment (PPE), such as a clean laboratory coat and appropriate gloves. Other essential tactics include avoiding mouth-pipetting, restricting speaking at the bench to only essential conversations, cleaning up any spills immediately, using pipettes to move any liquids (rather than pouring), and handling only one cell line at a time.

References

Boslett B, Nag S, Resnick A. Detection and Antibiotic Treatment of Mycoplasma arginini Contamination in a Mouse Epithelial Cell Line Restore Normal Cell Physiology. BioMed Res. Int. 2014. 532105

Drexler HG, Uphof CC. Comparative PCR analysis for detection of mycoplasma infections in continuous cell lines. In Vitro Cell. Dev. Biol. Animal 2002. 38:79–85

Drexler HG, Uphof CC. Mycoplasma contamination of cell cultures: Incidence, sources, effects, detection, elimination, prevention. Cytotechnology 2002. 39: 75–90

Olarerin-George AO, Hogenesch JB. Assessing the prevalence of mycoplasma contamination in cell culture via a survey of NCBI's RNA-seq archive. Nucleic Acids Research 2015. 43(5): 2535–2542

Rottem S, Kosower NS, Kornspan JD. Contamination of Tissue Cultures by Mycoplasmas. Biomedical Tissue Culture 2012. Book chapter 3, ISBN 978–953–51–0788–0

Uphoff CC, Meyer C, Drexler HG. Elimination of mycoplasma from leukemia–lymphoma cell lines using antibiotics. Leukemia 2002.16(2): 284–8

Young L, Sung J, Stacey G, Masters JR. Detection of Mycoplasma in cell cultures. Nature Protocols 2010. May;5(5):929-34