Mysterious mycoplasma – a question of culture?!

Firstly reported in 1956 (Robinson et al. 1956), the potential presence of mycoplasma in cell culture laboratories challenges scientists. The parasitic mycoplasmas represent a serious problem for all cell line-related fields in research as well as in industrial facilities for development or manufacture of cell-derived biological and pharmaceutical products, including vaccines, monoclonal antibodies, drugs, and products for gene and cell therapy. Still, there is no perceivable reduction of cell culture infection rates (Ryan 2008), even though risks and consequences caused by mycoplasma infections are known for decades, and strategies for their prevention, detection and elimination are well established. Why are so many cell lines – while commonly well fostered by their cell culturists – still insufficiently protected against the cell wall-free invader? Is this a cause of carelessness, or ­rather a lack of knowledge? Unfortunately we cannot provide any data regarding this question – but a lot of facts demonstrating the importance of the unpopular subject.

How do mycoplasmas commonly enter our labs and cultures?

Mycoplasmas are omnipresent; their broad range of hosts includes humans and other mammals, birds, reptiles, fish, insects and plants (Razin et al. 1998). However, in cell culture laboratories, 95% of all continuous cell line infections are caused by only six species originating from bovine (M. arginini ­& Acholeplasma laidlawii), swine (M. hyorhinis) – and human (M. orale, M. ­fermentans, M. hominis) (Drexler and Uphoff, 2002). The main source of mycoplasma contaminations today are my­co­plasma-infected cell cultures used in the same laboratory (Rottem & Barile 1993, Drexler et al. 2002; Drexler & Uphoff 2002). The infection may be transferred by aerosols, particulates and inadequate cell culture technique directly – or indirectly via media, solutions and ­laboratory equipment contaminated by previous use in processing mycoplasma-infected cells. As a result, 15–35% of all continuous cell lines are positive for mycoplasma, but only 1% of the primary cell cultures (Drexler and ­Uphoff, 2002). The second leading source is the laboratory personnel, explaining the fact that mycoplasma species from human are the most common contaminates ­(responsible for 40–80% of the infections) with M. orale, commonly colonizing the oral cavity, representing the primary ­species isolated from contaminated cell cultures.

Mycoplasma species from bovine or swine can be traced back to contaminated sera and other animal-derived products, e.g. the prevalent presence of A. laidlawii and M. arginini implicates fetal or newborn bovine serum as the primary source of infection.

By now, sera and media are rarely the source of mycoplasma contamination (Lincoln and Lundin 1990; Armstrong et al. 2009) as long as they are purchased from reputable manufacturers that sterilize their products by several filtration steps using a 0.1 µm pore membrane filter and frequently control sterility.

What makes mycoplasma species worse than other bacterial contaminates – and why is it a must to banish them from cell cultures?

In contrast to “common” bacteria, these ­tiny prokaryotes do not possess a cell wall. Together with other cell wall-lacking bacteria – species of ureaplasma, acholeplasma, anaeroplasma, spiroplasma - they form the class of mollicutes. Nevertheless, the terms “mycoplasma” or formerly “pleuropneumonia-like organisms (PPLO)” and “mollicutes” are often used synonymously. Due to the absence of a cell wall, mycoplasmas are unaffected by antibiotics that interfere with peptidoglycan formation, namely ­beta-lactam antibiotics. These include penicillin-derivatives, cephalosporins, and carbapenemes. Furthermore they are very flexible in shape which in addition to their small size (ranging from 0.1 to 0.8µm in diameter, depending on the literature) makes them difficult to filter from solutions. Mycoplasma species easily penetrate the membrane of 0.2m filters commonly used for sterilization of media, sera and other in-autoclavable reagents. Mycoplasma’s general dependence on complex enriched media (including host cell nutrients) and defined environmental conditions – both perfectly realized in cell culture – and their very slow growth rates complicate identification of infected cells by common microbiological cultivation methods. Their small size and missing cell wall allows them to achieve high densities in cell cultures; often without being detectable by turbidity, cytopathogenicity or even microscopic examination. However, the consequences of mycoplasma contaminations should not be underestimated; neither in regard to ­research (and the researcher’s career!), nor in terms of serious health risks for humans and animals. Please keep in mind that the mycoplasma family is composed of a number of pathogenic organisms!

By growing covertly and undisturbed within a cell culture, mycoplasma can easily take over the control of reagents, equipment, and other cell lines within weeks (McGarrity 1976). Be aware that the lack of visible effects provides a false sense of ­security: While often behaving inconspicuous at first glance, the fastidious organisms are able to influence nearly every single ­cellular function, ranging from a decelerated growth rate to metabolic (including protein, RNA, DNA synthesis) and morphologic changes. All these effects are mainly based on a competition for essential nutrients ­(nucleosides, nucleotides, nucleobases, arginine and other amino acids, fatty acids, sugars, etc.) and the release of toxic, cytolytic or acidic metabolites. By up- and down-regulation of cytokines and growth factors, stress-response genes, transport proteins, receptors, ion channels, oxidases, tumor supressors and oncogenes, mycoplasmas significantly alter the gene expression profiles of cultured cells (Miller et al. 2003). Therefore they make any experiment carried out with infected cells questionable! Furthermore they are known to cause chromosomal aberrations in vitro, with chromosomal breakage, translocation events, and reduction or augmentation in chromosome number being the most frequent outcomes. Virus propagation might also be influenced in both directions, positively (by inhibiting interferon induction and activity) as well as negatively (by competing for essential ­nutrients). Even though there are a large number of potential effects described in literature, it is unpredictable which effect will occur. Possible effects depend on mycoplasma species and strain, the infected cell type, and certainly on environmental conditions (Rottem & Barile 1993).

Finally, besides biosafety concerns, the consequences of mycoplasma contamination on laboratory work are loss of time, efforts, money (regarding cells, media, materials, but also valuable biopharmaceuticals if cultures were used for production of vaccines, antibodies or drugs) and good reputation. Research based on ­mycoplasma-contaminated cell lines will produce inaccurate or erroneous results yielding misleading publications. Consider the personal embarrassment and maybe the loss of good reputation if the published results are proven to be faulty due to a conta­mination problem. And how awkward will it be to get informed by a colleague that the cell line you provided him is contaminated? All these factors should be rethought when calculating to take the risk of covert mycoplasma infections by NOT testing cell cultures and NOT actively preventing them by good laboratory practise.

To sum up, a mycoplasma-free cell culture is PRECONDITION for safety and purity of cell-derived products and reliable results in scientific experiments.
The good message: it is possible to ­minimize the risk of general mycoplasma contaminations – and to exclude serious outbreaks.

How can I avoid contaminations?!

Probably, there will never be a point in time when mycoplasma contaminations are completely banished from our labs – as long as humans are working there. But carrying out some general principles will surely minimize the risk of contaminations and prevent costly or embarrassing situations.

// Strictly follow aseptic techniques and practices, including no unnecessary talking, no mouth pipetting, no media supply by pouring, regular hand washing and disinfection! Do not use the laminary flow for storage of solutions and equipment! Only work with ONE cell line at a time and use separate materials for each cell line to avoid cross-contaminations! Make sure all media, solutions and materials are properly sterilized – the same is true for any kind of occurring waste of course!

// Frequently clean and disinfect surfaces, laminar flows, incubators, water baths and all other equipment – before AND after the working procedure. Make sure the laboratory is cleaned up regularly and only authorized persons have access to the working area.

// Use antibiotics responsibly. For routine culture work antibiotic-free media should be employed. General usage of antibiotics to mask low hygiene levels, a lack of good aseptic techniques, or improper cell culture facilities is not a solution to the problem! Quite the contrary, non-responsible use of antibiotics will make the situation even worse.

// Isolate incoming cell cultures (use a separate incubator or at least sealed flasks as well as separate culture media and materials) until the mycoplasma test results are proven to be negative.

// Test frequently for contamination – ­regardless of whether the cell culture contains any antibiotics or not! Routine testings for the presence of mycoplasma species are an absolute must for the responsible scientist! Only by identification and treatment or elimination of the infected cell line the risk of further (cross-) contaminations is banished and experiments yield stable and reliable results.

Furthermore, it is highly recommended to freeze a cell stock as a backup for damaged or lost cell cultures. When dealing with cells of limited life span, cryopreservation is invaluable anyway. But also a stock of continuous cell lines should be stored properly below -130°C to prevent in vitro cellular alteration (Hughes et al. 2007; Stacey & Masters 2008) and maintain reliable cultures of consistent quality for research and biopharmaceutical production. The advantages of a cryopreserved cell bank are a reduced risk of (cross-) contamination with microorganisms or other cell lines, prevention of phenotypic or genotypic drifts, and damages due to cell aging. But caution: Please be aware that mycoplasmas are able to survive freezing in liquid nitrogen – even without cryopreservation. For that reason, a contaminated liquid nitrogen container (e.g. due to an inadequatly closed or contaminated sample of cells) might be a source of mycoplasma. But how do they enter the cell culture-containing cryo tube? Storage in the liquid phase together with a non-sufficient tube filling level might be the way in. Liquid nitrogen has the tendency to permeate the cryo tube, especially if it is filled insufficiently. To avoid any contamination risk, cryogenic vials should be properly stored in the vapour phase of the liquid nitrogen container.

How can I identify a mycoplasma contamination?

The most sensitive method to detect mycoplasma is the direct culture method in suitable broth and solid media to obtain visible colonies. Theoretically, a single CFU (colony forming unit) per sample volume is detectable. Unfortunately this method is also the most time consuming (up to 28 days; due to the slow growth of mycoplasma species), and it requires experienced personnel conducting the experiments under controlled environmental conditions. Even if the difficult procedure (to start with the complex medium composition often requiring non-standard adjustments for individual species up to analysis of the results) is properly conducted, the method is not 100% effective since some fastidious strains may not grow in pure culture. Therefore, an indirect detection method should be performed in addition. The most sensitive indirect mycoplasma tests are based on DNA fluorochrome staining (e.g. using DAPI) and PCR. Even if the detection limit of these methods is lower than for the direct culture method, they are absolutely sufficient for routine testings. Commonly, mycoplasma-contaminated cell cultures show high densities of mycoplasma (up to 107–108 CFU/ml) that are well suitable for the detection limits of these methods. In contrast to the PCR alternative, the traditional fluorescence staining method requires more time and experience. In addition, the DNA-binding fluorescent stain is cancerous and needs to be handled carefully. Hence, for routine mycoplasma screenings, PCR analysis is recommended (Drexler & Uphoff 2002). This method is sensitive (depending on the kit almost as sensitive as the direct culture method), very fast (results are obtained within hours) and detects cultivable as well as non-cultivable mycoplasma species. Furthermore, at least with commercially available kits, this method is very easy to perform and does not require a specific expertise.

What can I do to eliminate mycoplasma from an infected cell culture?

This answer is easy: Autoclave the conta­minated cells, at best, together with any bottle of medium and solution used with this relevant culture. Don’t forget subsequent cleaning and disinfection of surfaces, hoods, incubators, pipettors etc. – or better, the whole lab! Make sure that other cell lines are not infected as well! Cleaning up a contaminated culture with an anti-mycoplasma treatment is recommended only for very valuable or irreplaceable cultures, and if the potential source of mycoplasma was previously banished from the laboratory. Efforts for the attempted rescue are high and, until now, no universal mycoplasma-eliminating reagent is available. Antibiotic resistance, cytotoxicity, and a reduced viability of chronically or multiply infected cells may be reasons to prevent curing (Fleckenstein & Drexler 1996).

Despite assured resistances, the most reliable and efficient treatment of mycoplasma contaminations is the addition of suitable antibiotics, such as quinolones, tetracyclines and macrolides (Drexler & Uphoff 2002). In an experiment with 251 chronically mycoplasma-positive cell lines, treatment with ciprofloxacin provided healing-levels of 78%, with 15% of the cell cultures remaining contaminated due to resistance and 7% loss by cell death during the elimination procedure. The combination of tiamulin and minocycline even reached curing of 82% of all treated cell cultures, showing a lower resistance level (7%) but higher cytotoxicity (11% of the cell cultures died during the treatment) (data taken from Drexler & Uphoff 2002). Besides the traditional mycoplasma-eliminating agents Myco-1 & 2 (tiamulin and minocyclin) and Myco-3 (ciprofloxacin), AppliChem now offers a new solution for effective and permanent removal of mycoplasma species from cell culture: Myco-4 provides a broad spectrum of activity (including any type of mycoplasma, acholeplasma, spiroplasma and entomoplasma) combined with very low cytotoxicity and a low resistance risk due to an initial biophysical mode of action.

The “mycoplasma problem” is known for decades – why does it still exist?!

There are two main reasons why mycoplasma contaminations are not banished from cell culture laboratories yet: First, half of the researchers still do not test their cell cultures for mycoplasma (Ryan 2008) and second, there is a tendency to rely on antibiotics instead on good aseptic practices.

Even though cell culture experts agree that general use of antibiotics can increase the severity of contamination problems, the routine use of antibiotics in cell culture laboratories is still prevalent. Particularly mycoplasma contamination rates are much higher in cell lines grown in antibiotic-containing medium than in antibiotic-free cultures (Barile 1973). If microorganisms, bacteria or fungi, are accidentally brought into antibiotic-free culture medium, they will replicate non-inhibited, soon leading to visible indicators of contamination: turbidity, filamentary structures, color changes due to pH alteration. In contrast, the presence of antibiotics will prevent the microbial growth – maybe. Unfortunately there is no absolute guarantee that the added antibiotics act against the introduced microorganisms (probably a mixture of different species), and sooner or later the user will face some kind of resistance phenomenon. If the introduced germ is fully resistant to the antibiotic, it will hopefully rapidly overgrow the culture and become visible within a short period of time. If the introduced microorganism only shows a partial resistance the situation is worse. Due to the latent static level of partly resistant contaminations, the risk of cross contaminations and usage of the affected culture in experiments or bio-production should not be underestimated. This worst case is very likely if the invader belongs to the species of mycoplasma (e.g. brought into the culture through aerosol droplets from the mouth of the cell culturist), since most common antibiotics used in cell culture do not act on mycoplasma! Besides the beta-lactams being ineffective anyway, high resistance levels of mycoplasma against streptomycin (88%), kanamycin (73%), gentamicin (80%) and neomycin (86%) (Lundin & Lincoln 1994) were determined.

Apart from Barile's observation of strongly increased rates of mycoplasma contamination, morphological and functional changes are other disadvantages one has to take into account (Kuhlmann 1996) when using antibiotics on a routine basis. Anyhow, there exist useful applications of antibiotics in cell culture, e.g. within the first two weeks of primary culture. In order not to create new resistances due to inactivation of the antibiotic, the antibiotic-containing medium should be refreshed frequently.

As an alternative to classical cell culture antibiotics like penicillin-streptomycin, ­AppliChem provides a new product to ­prevent microbial growth in cell cultures: ­CellCultureGuard. This combination of ­selected antibiotics (one being a fluoroquinolone) offers a wide range of anti-microbial activity, making it our first choice cell culture reagent: CellCultureGuard is active against extra- and intracellular bacteria, mycoplasma, protozoa and fungi (yeast). Additionally, it is highly compatible with resistance markers and bears a low risk of resistance development.

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