Mycoplasma Contamination in Cell Culture: Causes, Problems and Solutions

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. laidlawii¹. 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.

Reference:

1) Drexler HG, Uphof CC (2002). In Vitro Cell. Dev. Biol. – Animal 38:79–85

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 methods, and PCR¹. Recently, 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 scattering².

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

Using PCR to Detect Mycoplasma Contamination

PCR is a powerful technique to amplify specific sequences of DNA in a sample and is a sensitive and rapid method for identifying mycoplasma contamination in your cell cultures. However, there is the risk of spreading the contamination from the positive control (or any of your samples that previously tested positive for Mycoplasma) to unaffected samples during the testing process, thereby leading to false positive results. While there are many PCR detection kits commercially available, many are not able to detect all species of Mycoplasma.

Culture-based Methods for Mycoplasma Detection

There are several standard culture methods available for the detection of Mycoplasma. These are:

1) The Broth and Agar Cultivation Procedure

This approach involves growing the contaminant in broth, plating the cells on nutrient agar plates, and then identifying if the contamination is due to Mycoplasma from the colonies formed. This method is highly specific and sensitive.

2) The Indicator Cell Culture Method

In this method, the bacterial contaminant is grown in a ‘test’ cell culture – a culture which is known to support high levels of mycoplasma proliferation. To identify if the contaminant is likely to be Mycoplasma, the bacterial DNA is fluorescently stained³. While highly sensitive, this technique has limited specificity for mycoplasma DNA.

3) The Sensor-cell Approach

It is possible to use specially engineered cells to detect the presence of Mycoplasma in cell cultures. These ‘sensor cells’ detect mycoplasma cells through their toll-like receptor (TLR) 2. In the presence of Mycoplasma species, this receptor is activated and triggers a signaling cascade, with the outcome being an observable color change. This is a highly sensitive method but has low specificity in comparison to other methods.

Manufactured and licensed biological products produced via cell substrates (e.g. viral vaccines, monoclonal antibodies, and similar products) need to be tested by methods (1) and (2) above to ensure the absence of mycoplasma contamination – this is a requirement for regulatory mycoplasma testing. A positive control with live mycoplasma bacteria and/or contaminated cells is also necessary in these assays. When the use of a positive control might compromise the sterility of a laboratory, it may be preferable to outsource the mycoplasma testing.

Biochemical Tests to Detect Mycoplasma Contamination

Mycoplasma species produce enzymes that are not found in eukaryotes. This makes it possible to detect them by looking for the presence of these enzymes in cell cultures. One kit designed for this is the MycoAlert^TM PLUS Mycoplasma Detection Kit, which contains the luciferase enzyme as a reporter. This method works as follows: first the mycoplasma cells are lysed, releasing their enzymes. These enzymes react with the substrate from the kit, which catalyzes the conversion of ADP to ATP. Luciferase uses this ATP to create a light signal, which can then be detected using a luminometer. This is a rapid method for identifying the presence of Mycoplasma species in cell cultures.

Using Fluorescence Microscopy to Detect Mycoplasma Contamination

It is also possible to detect mycoplasma DNA by fluorescence microscopy by using a non-specific DNA stain⁴. This stain is added to the culture medium and the presence of mycoplasma DNA can be confirmed by the appearance of bright foci/small clusters amongst the cells, when observed using a fluorescence microscope.

References:

1) Drexler HG, Uphof CC (2002). Cytotechnology 39: 75–902) Boslett et al. (2014). BioMed Research International, article ID 5321053) Young et al. (2010). Nature Protocols 5:5

2) Boslett et al. (2014). BioMed Research International, article ID 532105

3) Young et al. (2010). Nature Protocols 5:5

4) Rottem et al. (2012). Book chapter 3, book: Biomedical Tissue Culture, ISBN 978–953–51–0788–0

Discarding your contaminated cell cultures may seem like an obvious response to mycoplasma contamination. However, if you are using a culture that you can no longer obtain, or that is particularly valuable, it could be worth trying to eliminate the contamination. Keep in mind that treatment to get rid of Mycoplasma can take weeks, if not longer.

Elimination of mycoplasma contamination can be carried out by using specially formulated reagents, such as MycoZap^TM 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 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 filtration¹. 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, 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 observed¹.

If antibiotics 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).

Elimination of mycoplasma contamination can also be achieved by using specially formulated reagents, such as MycoZap^TM Mycoplasma Elimination Reagent. This reagent combines antibiotic and antimetabolic agents to eliminate detectable mycoplasma contamination in as little as four days. We have optimized it to clear mycoplasma contamination with minimal effects to your cell cultures.

References:

1) Uphoff et al. (2002). Leukemia 16: 284–288

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^TM Antibiotics, can be a way of preventing contamination.

3. Ensure You Adopt Aseptic Techniques

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:

1) Uphoff et al. (2002). Leukemia 16: 284–288