Mycoplasma contamination of cell cultures: Incidence, sources, effects, detection, elimination, prevention

The contamination of cell cultures by mycoplasmas remains a major problem in cell culture. Mycoplasmas can produce a virtually unlimited variety of effects in the cultures they infect. These organisms are resistant to most antibiotics commonly employed in cell cultures. Here we provide a concise overview of the current knowledge on: (1) the incidence and sources of mycoplasma contamination in cell cultures, the mycoplasma species most commonly detected in cell cultures, and the effects of mycoplasmas on the function and activities of infected cell cultures; (2) the various techniques available for the detection of mycoplasmas with particular emphasis on the most reliable detection methods; (3) the various methods available for the elimination of mycoplasmas highlighting antibiotic treatment; and (4) the recommended procedures and working protocols for the detection, elimination and prevention of mycoplasma contamination. The availability of accurate, sensitive and reliable detection methods and the application of robust and successful elimination methods provide powerful means for overcoming the problem of mycoplasma contamination in cell cultures. This revised version was published online in August 2006 with corrections to the Cover Date.     

Comparative antibiotic eradication of mycoplasma infections from continuous cell lines

Accumulating data implicate mycoplasma contamination as the single biggest problem in the culture of continuous cell lines. Mycoplasma infection can affect virtually every parameter and functional activity of the eukaryotic cells. A successful alternative to discarding infected cultures is to attempt to eliminate the contaminants by treatment with specific and efficient antimycoplasma antibiotics. The addition of antibiotics to the culture medium during a limited period of time (1-3 wk) is a simple, inexpensive, and very practical approach for decontaminating continuous cell lines. Here, we examined the effectiveness of several antibiotic treatment protocols that we have employed routinely in our cell lines bank. On an aggregate, 673 cultures from 236 chronically mycoplasma-positive cell lines were exposed to one of the following five antibiotic regimens: mycoplasma removal agent (quinolone; a 1-wk treatment), enrofloxacin (quinolone; 1 wk), sparfloxacin (quinolone; 1 wk), ciprofloxacin (quinolone; 2 wk), and BM-Cyclin (alternating tiamulin and minocycline; 3 wk). The mycoplasma infection was permanently (as determined by three solid mycoplasma detection assays) eliminated by the various antibiotics in 66-85% of the cultures treated. Mycoplasma resistance was seen in 7-21%, and loss of the culture as a result of cytotoxically caused cell death occurred in 3-11% of the cultures treated. Overall, 223 of the 236 mycoplasma-positive cell lines could be cured in a first round of antibiotic treatment with at least one regimen. Taken together, 95% of the mycoplasma-infected cell lines were permanently cleansed of the contaminants by antibiotic treatment, which validates this approach as an efficient and technically simple mycoplasma eradication method.     

In situ detection of mycoplasma contamination in cell cultures by fluorescent Hoechst 33258 stain

A simple, fast, and easily reproducible routine laboratory technique for detecting mycoplasma contamination in cell cultures is reported. Cells grown on a coverslip are fixed directly with Carnoy's, air-dried, stained with DNA-specific fluorescent Hoechst 33258, and examined microscopically. All cultures that were infected with mycoplasmas had readily discernible, small, morphologically uniform, bright fluorescent bodies in the extranuclear and intercellular space in contrast to the non-contaminated control cultures in which the extra-nuclear background appeared uniformly dark. To probe the degree of sensitivity to detect mycoplasmas, control cultures were infected with aliquots from serially diluted cells or media collected from Mycoplasma hyorhinus infected cultures. The lowest infection rate (0.40% by sampling 1 000 cells in average per culture 4–24 h after infection) scored presently, however, can easily be lowered by increasing sample size since a cell infected with even one mycoplasma can be discerned. These mycoplasmas resisted centrifugation at 2 500 rpm for 30 min and easily filtered through 0.22 μm pore-size filter membrane. Amazingly infection rate of 0.63% scored from 24 h post-infection incubation attained 100% contamination with several hundreds of mycoplasmas per host cell within 120 h.     

Comparative PCR analysis for detection of mycoplasma infections in continuous cell lines

Mycoplasma contamination of cell lines is one of the major problems in cell culturing. About 15-35% of all cell lines are infected with a limited number of mycoplasma species of predominantly human, swine, or bovine origin. We examined the mycoplasma contamination status in 495 cell cultures by polymerase chain reaction (PCR) assay, microbiological culture method, and deoxyribonucleic acid-ribonucleic acid (DNA-RNA) hybridization, and in 103 cell cultures by PCR and DNA-RNA hybridization, in order to determine the sensitivity and specificity of the PCR assay in routine cell culture. For those two cohorts, results for the three or two assays were concordant in 92 and 91% of the cases, respectively. The sensitivity (detection of true positives) of this PCR detection assay was 86%, and the specificity (detection of true negatives) was 93%, with positive and negative predictive values (probability of correct results) of 73 and 97%, respectively. PCR defined the mycoplasma status with 92% accuracy (detection of true positives and true negatives). The mycoplasma contaminants were speciated by analyzing the PCR amplification fragment using several restriction enzymes. Most of the cultures (47%) were infected with Mycoplasma fermentans, followed by M. hyorhinis (19%), M. orale (10%), M. arginini (9%), Acholeplasma laidlawii (6%), and M. hominis (3%). To sum up, PCR represents a sensitive, specific, accurate, inexpensive, and quick mycoplasma detection assay that is suitable for the routine screening of cell cultures.     

Real-time PCR assay is superior to other methods for the detection of mycoplasma contamination in the cell lines of the National Cell Bank of Iran

Mycoplasmas are the most important contaminants of cell cultures throughout the world. They are considered as a major problem in biological studies and biopharmaceutical economic issues. In this study, our aim was to find the best standard technique as a rapid method with high sensitivity, specificity and accuracy for the detection of mycoplasma contamination in the cell lines of the National Cell Bank of Iran. Thirty cell lines suspected to mycoplasma contamination were evaluated by five different techniques including microbial culture, indirect DNA DAPI staining, enzymatic mycoalert^® assay, conventional PCR and real-time PCR. Five mycoplasma-contaminated cell lines were assigned as positive controls and five mycoplasma-free cell lines as negative controls. The enzymatic method was performed using the mycoalert^® mycoplasma detection kit. Real-time PCR technique was conducted by PromoKine diagnostic kits. In the conventional PCR method, mycoplasma genus-specific primers were designed to analyze the sequences based on a fixed and common region on 16S ribosomal RNA with PCR product size of 425 bp. Mycoplasma contamination was observed in 60, 56.66, 53.33, 46.66 and 33.33 % of 30 different cell cultures by real-time PCR, PCR, enzymatic mycoalert^®, indirect DNA DAPI staining and microbial culture methods, respectively. The analysis of the results of the different methods showed that the real-time PCR assay was superior the other methods with the sensitivity, specificity, accuracy, predictive value of positive and negative results of 100 %. These values were 94.44, 100, 96.77, 100 and 92.85 % for the conventional PCR method, respectively. Therefore, this study showed that real-time PCR and PCR assays based on the common sequences in the 16S ribosomal RNA are reliable methods with high sensitivity, specificity and accuracy for detection of mycoplasma contamination in cell cultures and other biological products.     

Efficiency of Plasmocin™ on various mammalian cell lines infected by mollicutes in comparison with commonly used antibiotics in cell culture: a local experience

Mycoplasma contamination is a deleterious event for cell culture laboratories. Plasmocin™ is used to prevent and eradicate mycoplasma infections from cell. In this study, 80 different mammalian cell lines from various sources; human, monkey, mice, hamster and rat were used to study and evaluate plasmocin™ efficiency and compare it to commonly used antibiotics such as BM-cyclin, ciprofloxacin and mycoplasma removal agent (MRA). It was shown that mycoplasma infections were eradicated by plasmocin™, BM-cyclin, ciprofloxacin and MRA in 65%, 66.25%, 20%, and 31.25%, respectively, of infected cell cultures. However, re-infection with mycoplasmas after the period of 4 months occurred in 10–80% of the studied cell lines. Cell cytotoxicity and culture death was observed in 25, 17.5 and 10% of the treated cells, for plasmocin™, BM-cyclin and MRA, respectively. In this study, Plasmocin™ showed strong ability to eradicate mollicutes from our cell lines with minimal percentage of regrowth. However, due to its high cell cytotoxicity it should be used with caution especially when dealing with expensive or hard-to-obtain cell lines. Amongst the antibiotics tested, BM-cyclin was shown to remove mycoplasma with the highest efficiency.     

Optimized PCR-based Detection of Mycoplasma

The maintenance of contamination-free cell lines is essential to cell-based research. Among the biggest contaminant concerns are mycoplasma contamination. Although mycoplasma do not usually kill contaminated cells, they are difficult to detect and can cause a variety of effects on cultured cells, including altered metabolism, slowed proliferation and chromosomal aberrations. In short, mycoplasma contamination compromises the value of those cell lines in providing accurate data for life science research.The sources of mycoplasma contamination in the laboratory are very challenging to completely control. As certain mycoplasma species are found on human skin, they can be introduced through poor aseptic technique. Additionally, they can come from contaminated supplements such as fetal bovine serum, and most importantly from other contaminated cell cultures. Once mycoplasma contaminates a culture, it can quickly spread to contaminate other areas of the lab. Strict adherence to good laboratory practices such as good aseptic technique are key, and routine testing for mycoplasma is highly recommended for successful control of mycoplasma contamination.PCR-based detection of mycoplasma has become a very popular method for routine cell line maintenance. PCR-based detection methods are highly sensitive and can provide rapid results, which allows researchers to respond quickly to isolate and eliminate contamination once it is detected in comparison to the time required using microbiological techniques. The LookOut Mycoplasma PCR Detection Kit is highly sensitive, with a detection limit of only 2 genomes per μl. Taking advantage of the highly specific JumpStart Taq DNA Polymerase and a proprietary primer design, false positives are greatly reduced. The convenient 8-tube format, strips pre-coated with dNTPs, and associated primers helps increase the throughput to meet the needs of customers with larger collections of cell lines.Given the extreme sensitivity of the kit, great care must be taken to prevent inadvertent contamination of samples and reagents. The step-by-step protocol we demonstrate highlights the precautions and practices required for reliable mycoplasma detection. We also show and discuss typical results and their interpretation. Our goal is to ensure the success of researchers using the LookOut Mycoplasma PCR Detection Kit.Download video file.^{(36M, mov)}     

细胞培养中支原体污染的PCR检测

根据支原体16s rDNA序列,选择RemyTeyssou设计的三条寡核苷酸链,组成两套引物:P_(1-2a)能检测出细胞培养中常见的各种支原体,P_(1-2b)能检出无胆甾原体。反应可检出体系中10CFV的菌体。此法先用于对实验室人为污染支原体Vero细胞的检测,后与DNA 染色法和培养法比较,检测了49份生物样品,其中24份传代细胞,PCR检测的阳性率为58％,DNA染色法为42％,培养法为33％;三者的灵敏性比较,PCR可检出10~(-3)稀释度的阳性样品,高于其他两种方法。此PCR方法快速、灵敏、特异,适用于细胞培养中支原体污染的检测。     

Assessing the prevalence of mycoplasma contamination in cell culture via a survey of NCBI's RNA-seq archive

Mycoplasmas are notorious contaminants of cell culture and can have profound effects on host cell biology by depriving cells of nutrients and inducing global changes in gene expression. Over the last two decades, sentinel testing has revealed wide-ranging contamination rates in mammalian culture. To obtain an unbiased assessment from hundreds of labs, we analyzed sequence data from 9395 rodent and primate samples from 884 series in the NCBI Sequence Read Archive. We found 11% of these series were contaminated (defined as ≥100 reads/million mapping to mycoplasma in one or more samples). Ninety percent of mycoplasma-mapped reads aligned to ribosomal RNA. This was unexpected given 37% of contaminated series used poly(A)-selection for mRNA enrichment. Lastly, we examined the relationship between mycoplasma contamination and host gene expression in a single cell RNA-seq dataset and found 61 host genes (P < 0.001) were significantly associated with mycoplasma-mapped read counts. In all, this study suggests mycoplasma contamination is still prevalent today and poses substantial risk to research quality.     

Prolonged hyperthermia eliminates mycoplasma from cultured human and rat brain tumor cell lines

Mycoplasma infection of mammalian cells in culture is a common occurrence that can affect the results of experimental protocols. Current methods of eliminating mycoplasma from cell cultures are usually tedious, time-consuming, and sometimes unsuccessful. In the present study, four cultured brain tumor cell lines (human U-251 MG, U-87 MG, SF-126, and rat 9L) were heavily contaminated with Mycoplasma orale. Heating the cultures to 41 degrees C for at least 96 h eliminated the contamination for up to 7 months, the maximum period of observation. The time chosen to assay for the presence of mycoplasma in cultures was critical: in some cultures heated for less than 96 h that initially appeared to be free of contamination, mycoplasma began to appear after 2 weeks. Heat-treated cells grew at the same rate as unheated control cells. Infected cells were more sensitive to X rays than uncontaminated cells, but the sensitivity reverted to normal after mycoplasma was eliminated by hyperthermia. The heating method does not require a cell cloning procedure or the use of exogenous materials. Treated cell cultures exhibit normal growth and radiation sensitivity, and the technique seems to be reliable and efficient.     

Virus contaminations of cell cultures – A biotechnological view

In contrast to contamination by microbes and mycoplasma, which can be relatively easily detected, viral contamination present a serious threat because of the difficulty in detecting some viruses and the lack of effective methods of treating infected cell cultures. While some viruses are capable of causing morphological changes to infected cells (e.g. cytopathic effect) which are detectable by microscopy some viral contaminations result in the integration of the viral genome as provirus, this causes no visual evidence, by means of modification of the cellular morphology. Virus production from such cell lines, are potentially dangerous for other cell cultures (in research labs)by cross contaminations, or for operators and patients (in the case of the production of injectable biologicals) because of potential infection. The only way to keep cell cultures for research, development, and the biotech industry virus-free is the prevention of such contaminations. Cell cultures can become contaminated by the following means: firstly, they may already be contaminated as primary cultures (because the source of the cells was already infected), secondly, they were contaminated due to the use of contaminated raw materials, or thirdly, they were contaminated via an animal passage. This overview describes the problems and risks associated with viral contaminations in animal cell culture, describes the origins of these contaminations as well as the most important virsuses associated with viral contaminations in cell culture. In addition, ways to prevent viral contaminations as well as measures undertaken to avoid and assess risks for viral contaminations as performed in the biotech industry are briefly described. This revised version was published online in August 2006 with corrections to the Cover Date.     

Mycoplasma contamination of leukemic cell lines alters protein expression determined by reverse phase protein arrays

Mycoplasma contamination is a major problem in cell culturing, potentially altering the results of cell line-based experiments in largely uncharacterized ways. To define the consequences of mycoplasma infection at the level of protein expression we utilized the reverse phase protein array technology to analyze the expression of 235 proteins in mycoplasma infected, uninfected post treatment, and never-infected leukemic cell lines. Overall, protein profiles of cultured cells remained relatively stable after mycoplasma infection. However, paired comparisons for individual proteins identified that 18.7% of the proteins significantly changed between the infected and the never-infected cell line samples, and that 14.0% of the proteins significantly altered between the infected and the post treatment samples. Six percent of the proteins were affected in the post treatment samples compared to the never-infected samples, and 7.2% compared to treated cells that had never had mycoplasma infection before. Proteins that were significantly altered in the infected cells were enriched for apoptotic signaling processes and auto-phosphorylation, suggesting an increased cellular stress and a decreased growth rate. In conclusion, this study shows that mycoplasma infection of leukemic cell lines alters the proteins expression levels, potentially confounding experimental results. This reinforces the need for regular testing of mycoplasma.     

Semi-automated relative quantification of cell culture contamination with mycoplasma by Photoshop-based image analysis on immunofluorescence preparations

Mycoplasma contamination in cell culture is a serious setback for the cell-culturist. The experiments undertaken using contaminated cell cultures are known to yield unreliable or false results due to various morphological, biochemical and genetic effects. Earlier surveys revealed incidences of mycoplasma contamination in cell cultures to range from 15 to 80%. Out of a vast array of methods for detecting mycoplasma in cell culture, the cytological methods directly demonstrate the contaminating organism present in association with the cultured cells. In this investigation, we report the adoption of a cytological immunofluorescence assay (IFA), in an attempt to obtain a semi-automated relative quantification of contamination by employing the user-friendly Photoshop-based image analysis. The study performed on 77 cell cultures randomly collected from various laboratories revealed mycoplasma contamination in 18 cell cultures simultaneously by IFA and Hoechst DNA fluorochrome staining methods. It was observed that the Photoshop-based image analysis on IFA stained slides was very valuable as a sensitive tool in providing quantitative assessment on the extent of contamination both per se and in comparison to cellularity of cell cultures. The technique could be useful in estimating the efficacy of anti-mycoplasma agents during decontaminating measures.     

Cross comparison of rapid mycoplasma detection platforms

The use of animal and plant derived raw materials in mammalian cell culture processes may provide a possible route of entry for adventitious contaminants such as mycoplasma. Mycoplasma contaminations of cell culture represent a serious challenge to the production of biotechnology derived therapeutics. The slow growing nature of mycoplasma can disguise their infection of cultures since cells may continue to proliferate, though at reduced levels and with lesser output of engineered protein. Rapid identification of mycoplasma contaminated cell cultures and materials enables a faster response time to prevent the spread of the contamination. We describe here the comparison of different mycoplasma detection methods: two nucleic acid-based technologies, the standard mycoplasma culture procedure, and a hybrid culture-quantitative PCR assay. In this study, a cell line infected with two species of mycoplasma was used to compare the different detection methods. Our data demonstrates that the two nucleic acid-based techniques are robust methods for detection of mycoplasma and have similar detection capability. In contrast, no mycoplasma was detected in the standard culture assay or in a hybrid culture-quantitative PCR assay. This shows a potential limitation of the culture assay that relies on the ability of mycoplasma to grow in broth media.     

The Effects of Mycoplasma Contamination upon the Ability to Form Bioengineered 3D Kidney Cysts

Mycoplasma contamination of cell cultures is a pervasive, often undiagnosed and ignored problem in many laboratories that can result in reduced cell proliferation and changes in gene expression. Unless contamination is specifically suspected, it is often undetected in two dimensional (2D) cultures and the resulting effects of mycoplasma contamination are rarely appreciated and can lead to incorrect conclusions. Three dimensional (3D) tissue cultures are increasingly utilized to explore tissue development and phenotype. However, 3D cultures are more complex than 2D cell cultures and require a more controlled cellular environment in order to generate structures necessary to mimic in vivo responses and are often maintained for longer time periods. Changes to the microenvironment are assumed to have a more extreme effect upon the success of 3D tissue cultures than 2D cell cultures, but the effects of mycoplasma have not been studied. To test this hypothesis, we grew 2D cell cultures and 3D tissues from pig kidney epithelial cells (LLC-PK1) that were contaminated with mycoplasma and the same stock of cells after mycoplasma removal. We did not observe an effect of mycoplasma contamination on proliferation in 2D monolayer cell culture. However, cyst formation in 3D tissues was altered, with effects upon the number, size and structure of cysts formed. These data serve to reinforce the necessity of testing cell stocks for mycoplasma contamination.     

Mycoplasma contamination of cell cultures: methods for its detection and the possible routes of Mycoplasma infection spread

83 continuous cell lines were screened for mycoplasma contamination by three methods: microbiological seeding for enriched media, staining with Hoechst-33258 stain, and autoradiography with 3H-thymidine incorporation. It has been shown that combination of different methods is necessary for the strict control of mycoplasma-contamination in cell cultures. Simultaneous manipulation with mycoplasma infected and pure cell lines leads to cross-contamination in 2-3 passages. Precautions are described to preclude mycoplasma-contamination during prolonged cell cultivation.     

Sensitivity of biochemical test in comparison with other methods for the detection of mycoplasma contamination in human and animal cell lines stored in the National Cell Bank of Iran

Mycoplasma contamination in cell culture is considered as serious problem in the manufacturing of biological products. Our goal in this research is to find the best standard and rapid method with high sensitivity, specificity, accuracy and predictive values of positive and negative results for detection of mycoplasma contamination in cell cultures of the National Cell Bank of Iran. In this study, 40 cell lines suspected to mycoplasma contamination were evaluated by three different methods: microbial culture, enzymatic mycoalert^® and molecular. Enzymatic evaluation was performed using the mycoalert^® kit while in the molecular technique, a universal primer pair was designed based on the common and fixed 16SrRNA ribosomal sequences used. Mycoplasma contaminations in cell cultures with molecular, enzymatic and microbial culture methods were determined as 57.5, 52.5 and 40 %, respectively. These results confirmed the higher rate of sensitivity, specificity and accuracy for the molecular method in comparison with enzymatic and microbial methods. Polymerase chain reaction (PCR) assay based on fixed and common sequences in the 16SrRNA, is a useful valuable and reliable technique with high sensitivity, specificity and accuracy for detection of mycoplasma contamination in cell cultures and other biological products. The enzymatic mycoalert^® method can be considered as a substitution for conventional microbial culture and DNA staining fluorochrome methods due to its higher sensitivity, specificity and speed of detection (<20 min).     

Treatment of mycoplasma contamination in a large panel of cell cultures

Summary Mycoplasmal contamination remains a significant impediment to the culture of eukaryotic cells. For certain cultures, attempts to eliminate the infection are feasible alternatives to the normally recommended disposal of the contaminated culture. Here, three antibiotic regimens for mycoplasmal decontamination were compared in a large panel of naturally infected cultures: a 1-wk treatment with the fluoroquinolone mycoplasma removal agent (MRA), a 2-wk treatment with the fluoroquinolone ciprofloxacin, and three rounds of a sequential 1-wk treatment with BM-Cyclin containing tiamulin and minocyclin. These antibiotic treatments had a high efficiency of permanent cure: MRA 69%, ciprofloxacin 75%, BM-Cyclin 87%. Resistance to mycoplasma eradication was observed in some cell cultures: BM-Cyclin 0%, MRA 20%, ciprofloxacin 20%. Nearly all resistant contaminants that could be identified belonged to the speciesMycoplasma arginini andM. orale. Detrimental effects of the antibiotics were seen in the form of culture death caused by cytotoxicity (in 5 to 13% of the cultures). Alterations of the cellular phenotypic features or selective clonal outgrowth might represent further untoward side effects of exposure to these antibiotics. Overall, antibiotic decontamination of mycoplasmas is an efficient, inexpensive, reliable, and simple method: 150/200 (75%) chronically and heavily contaminated cultures were cured and 50/200 (25%) cultures could not be cleansed and were either lost or remained infected. It is concluded that eukaryotic cell cultures containing mycoplasmas are amenable to antibiotic treatment and that a cure rate of three-quarters is a reasonable expectation.     

Degradation of nuclease-stabilized RNA oligonucleotides in Mycoplasma-contaminated cell culture media

Artificial RNA reagents such as small interfering RNAs (siRNAs) and aptamers often must be chemically modified for optimal effectiveness in environments that include ribonucleases. Mycoplasmas are common bacterial contaminants of mammalian cell cultures that are known to produce ribonucleases. Here we describe the rapid degradation of nuclease-stabilized RNA oligonucleotides in a human embryonic kidney 293 (HEK) cell culture contaminated with Mycoplasma fermentans, a common species of mycoplasma. RNA with 2'-fluoro- or 2'-O-methyl- modified pyrimidines was readily degraded in conditioned media from this culture, but was stable in conditioned media from uncontaminated HEK cells. RNA completely modified with 2'-O-methyls was not degraded in the mycoplasma-contaminated media. RNA zymogram analysis of conditioned culture media and material centrifuged from the media revealed several distinct protein bands (ranging from 30 to 68?kDa) capable of degrading RNA with 2'-fluoro- or 2'-O-methyl-modified pyrimidines. Finally, the mycoplasma-associated nuclease was detected in material centrifuged from the contaminated culture supernatants in as little as 15 minutes with an RNA oligo-containing 2'-O-methyl-modified pyrimidines and labeled with a 5'-fluorescein amidite (FAM) and 3'-quencher. These results suggest that mycoplasma contamination may be a critical confounding variable for cell culture experiments involving RNA-based reagents, with particular relevance for applications involving naked RNA (e.g., aptamer-siRNA chimeras).