In the story of cell culture, Mycoplasma is the clear antagonist. These tiny, malleable bacteria lack a rigid cell wall and are notoriously difficult to treat with antibiotics. Given their prevalence, dealing with Mycoplasma contamination in cell culture is, unfortunately, an all-too-common experience.

Dealing with Mycoplasma contaminated cell cultures involves discarding cultures or using a strategy to treat your cultures. Treatment is time-consuming and should only be done in certain cases.

Let’s look at the ways to deal with Mycoplasma contamination, and how to help prevent future contaminating events.

There are a few different options for dealing with cells once they’ve been contaminated. Even better, though, is to take steps to prevent contamination before it starts.

·         Discard standard cell lines

·         Treat valuable or irreplaceable cells

·         Deep clean the cell culture room

·         Screen incoming cell lines

 

Article Table of Contents:

Discard standard cell lines

How to treat valuable or irreplaceable cells

Deep clean the cell culture room

Screen incoming cell lines

References

Discard standard cell lines

If you’re working with a standard cell line, then you should just throw away the contaminated cells and start over. Examples of this include cell lines that you can easily order from a vendor, or cell lines for which you have frozen, uncontaminated aliquots that you could quickly start over with.

The process for properly discarding your Mycoplasma contaminated cells is simple. Just bleach your contaminated cultures and discard them responsibly.

But let’s say these are not standard cells. They’re valuable and hard to replace. Fortunately, there is a way to try to salvage important or tough to source cells. However, the time required for this process, and the uncertainty that it introduces to your cells and downstream experiments make it only worth the effort for the most valuable or difficult to replace cells.

How to treat valuable or irreplaceable cells

Some cells are inherently more valuable or difficult to source. Primary cells that were recently removed from their biological context are an example of this.

One source of these is primary tumor samples that are removed from patients (with consent) during biopsies or surgery and are used to establish cell lines to better understand that form of cancer.

You can’t just pick up the phone (or go to a website) to order more of these. You have to wait for the next biopsy or surgery, and even then, those cells will be from a different patient and will likely be fundamentally different.

So, these types of samples are incredibly valuable, and a limited resource that is tough, if not impossible to replace. If you have this type of sample, you can try using antibiotics to kill the Mycoplasma and get the culture back to only the desired type of cells.

The problem is that not all antibiotics work well on Mycoplasma. Typically, however, ciprofloxacin or tetracycline/doxycycline are recommended for treating Mycoplasma. If any of these antibiotics appear to work for your contaminated sample, you’ll want to then grow your cells without antibiotic for at least two weeks afterward to make sure that the Mycoplasma isn’t still present (Figure 1) (Uphoff et al, 2012).

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Figure 1. Treatment timeline for valuable cells that are contaminated with Mycoplasma.

 

Ideally once you remove the antibiotic, the Mycoplasma doesn’t return and your cells of interest grow just fine. Antibiotics have side effects on mammalian cells and typically aren’t recommended for long-term culture. So, washing out the antibiotic should help to reset the cells to their unperturbed state.

When attempting to treat contaminated cells, you’ll want to document the following:

• The contamination
• What was done to treat the contamination

Then you will want to try to benchmark the treated cells and compare back to before they were infected.

If there is a standard assay that you’ve been using these cells for, then grow up a batch of the treated cells and test them out in this assay. Examples could include growth assays, luciferase reporter assays, gene expression, metabolism, really anything that you had measured for these cells before contamination.

Once you retest your cells, if the results are comparable to the pre-contaminated cells, then you will be more confident in moving forward with this antibiotic-treated batch.

Deep clean the cell culture room

Besides dealing with the contaminated cells, you will also want to address the lab environment to try to eliminate any potential sources of contamination. In addition to being introduced through new and contaminated cell lines, Mycoplasma can come from:

• Nonsterile supplies
• Nonsterile media and solutions
• Scientists and dirty lab coats
• Lab surfaces such as incubators, liquid nitrogen tanks, and cell culture hoods

Even during routine cell culture work, it is important to work with sterile equipment, routinely clean your workspace, and use clean or disposable lab coats. If you’re having contamination issues, then it is worth doing a deep clean of the entire lab space and doubling down on your efforts for proper aseptic technique.

While it might take a few hours out of your day to perform a deep clean, that sure beats losing weeks having to treat or grow up new cells and repeating experiments over again due to contamination.   

 

Screen incoming cell lines

Dealing with Mycoplasma can be quite labor- and time-intensive. It’s much better to prevent contamination than to deal with it once it starts. In addition to routine cleaning and proper aseptic technique, screening any new incoming cell lines for Mycoplasma is one of the best ways to prevent contamination.

There are a number of ways to screen for Mycoplasma. One of the best, and most frequently used ways is PCR. You can do PCR yourself with primers that recognize over 90% of Mycoplasma species (Siegl et al, 2023).

Briefly, that includes, lysing cells from the line of interest, isolating the DNA, performing PCR, then analyzing the resulting samples on an agarose gel (Figure 2).

Figure 2. PCR samples can be analyzed on an agarose gel. Lane 2 shows a positive result as the upper band and a control band (lower). Lane 3 shows up negative as no upper band is present while only the control band is present.

 

When using PCR to test for Mycoplasma, there will be a Mycoplasma-specific set of primers corresponding to the upper band on the agarose gel (Figure 3, right) and a control set of primers to make sure that the PCR worked corresponding to the lower band on the agarose gel.

Additionally, there are commercial services you can send a sample of your cell line to and pay them to test for Mycoplasma contamination.

You will want to make sure that Mycoplasma tests have been performed whenever you purchase new cell lines. And if you’re gifted a cell line from a collaborator, you will definitely want to pay for Mycoplasma testing or do it yourself.

It is also really important to quarantine new cell lines from all of your existing cell lines until you’re sure that they are Mycoplasma free.

By the way, testing for Mycoplasma contamination doesn’t have to be limited to when you receive new cell lines. Recommendations for routine testing of Mycoplasma range from every six months for basic research to every two weeks for clinical samples. How often you test for Mycoplasma will probably depend on the exact nature of your studies, and how often your lab is facing contamination issues. However, if your samples are suddenly giving unusual results then it probably would be worthwhile to retest for Mycoplasma.

When it comes to mammalian cell culture, it really is worth the time and effort to be proactive in preventing Mycoplasma contamination. Follow the steps in this article to help prevent Mycoplasma infection, and to resolve it if your cells do get contaminated. GoldBio provides high-quality and affordable reagents to help treat and test for Mycoplasma including antibiotics, PCR master mix, and agarose gel components, so check those links out below.

 

References

DesRochers, T. M., Kuo, I. Y., Kimmerling, E. P., Ehrlich, B. E., & Kaplan, D. L. (2015). The effects of Mycoplasma contamination upon the ability to form bioengineered 3D kidney cysts. PloS one, 10(3), e0120097. https://doi.org/10.1371/journal.pone.0120097

Gautier-Bouchardon A. V. (2018). Antimicrobial Resistance in Mycoplasma spp. Microbiology spectrum, 6(4), 10.1128/microbiolspec.arba-0030-2018. https://doi.org/10.1128/microbiolspec.ARBA-0030-2018

Gong, H., Zölzer, F., von Recklinghausen, G., Rössler, J., Breit, S., Havers, W., Fotsis, T., & Schweigerer, L. (1999). Arginine deiminase inhibits cell proliferation by arresting cell cycle and inducing apoptosis. Biochemical and biophysical research communications, 261(1), 10–14. https://doi.org/10.1006/bbrc.1999.1004

Miller, C. J., Kassem, H. S., Pepper, S. D., Hey, Y., Ward, T. H., & Margison, G. P. (2003). Mycoplasma infection significantly alters microarray gene expression profiles. BioTechniques, 35(4), 812–814. https://doi.org/10.2144/03354mt02

Nikfarjam, L., & Farzaneh, P. (2012). Prevention and detection of Mycoplasma contamination in cell culture. Cell journal, 13(4), 203–212.

Olarerin-George, A. O., & Hogenesch, J. B. (2015). Assessing the prevalence of Mycoplasma contamination in cell culture via a survey of NCBI's RNA-seq archive. Nucleic acids research, 43(5), 2535–2542. https://doi.org/10.1093/nar/gkv136

Shimizu, T., Kida, Y., & Kuwano, K. (2005). A dipalmitoylated lipoprotein from Mycoplasma pneumoniae activates NF-kappa B through TLR1, TLR2, and TLR6. Journal of immunology (Baltimore, Md. : 1950), 175(7), 4641–4646. https://doi.org/10.4049/jimmunol.175.7.4641

Siegl, D., Kruchem, M., Jansky, S., Eichler, E., Thies, D., Hartwig, U., Schuppan, D., & Bockamp, E. (2023). A PCR protocol to establish standards for routine Mycoplasma testing that by design detects over ninety percent of all known Mycoplasma species. iScience, 26(5), 106724. https://doi.org/10.1016/j.isci.2023.106724

UNC Lineberger Comprehensive Cancer Center. (2019, March 1). Mycoplasma. https://unclineberger.org/tissueculture/contaminant/Mycoplasmacontam/

Uphoff, C. C., Denkmann, S. A., & Drexler, H. G. (2012). Treatment of Mycoplasma contamination in cell cultures with Plasmocin. Journal of biomedicine & biotechnology, 2012, 267678. https://doi.org/10.1155/2012/267678

 

 

 

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antibiotics cell culture mycoplasma Simon Currie

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