Contamination of Tissue Cultures by Mycoplasmas

Rottem, Shlomo; Kosower, Nechama S.; Kornspan, David

doi:10.5772/51518

Cited by 22 publications

(44 citation statements)

References 116 publications

(138 reference statements)

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“…Es ist daher kaum verwunderlich, dass diese Bakterien die "idealen" Kontaminanten von Zellkulturen und zellulärem Ausgangsmaterial darstellen. Es ist zudem nicht möglich, Mykoplasmen mit konventionellen 0,2 µm Sterilfiltersystemen zurückzuhalten oder diese zuverlässig in der klassischen Sterilitätstestung nachzuweisen [37,38]. Hinzu kommt, dass durch das Fehlen der Peptidoglykan-basierten Zellwand in der Zellkultur eingesetzte Antibiotika, wie Penicillin und dessen Analoge, gegen diese Bakterien unwirksam sind.…”

Section: Mykoplasmenunclassified

“…Häu-fig wird die direkte Auswirkung einer Kontamination mit Mykoplasmen auf das Produkt selbst übersehen: Aus der Literatur ist bekannt, das Mykoplasmen die Funktion von Zellen verändern können, d. h. möglich ist sowohl ein verändertes Genexpressionsmuster und Proteinprofil als auch letztendlich eine veränderte Zellaktivität. Es ist zudem nicht ausgeschlossen, dass diese Veränderungen in seltenen Fällen in einer bösartigen Transformation der eukaryotischen Zellen und damit in der Ausbildung von Tumoren resultieren können [38][39][40].…”

Section: Mykoplasmenunclassified

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Aktuelles Konzept zur mikrobiologischen Sicherheit von zellbasierten Arzneimitteln

Schurig

Karo

Sicker

et al. 2015

Bundesgesundheitsbl.

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Ensuring microbiological safety in advanced-therapy medicinal products is still a big challenge for manufacturers. There are fundamental problems, especially in cell-based medicinal products, regarding sterility of source materials, short shelf-life of final products, and the selection of suitable microbiological methods. Different from classical medicinal products, there is the need to evaluate a large number of possible risks and to calculate the risk-benefit balance. Depending on the source material, the presence of micro-organisms with specific growth requirements has to be considered. They cannot be detected by conventional testing methods, but may replicate after the application of the preparation in the recipient. Mycoplasmas are the primary representatives of these contaminants and specific testing procedures are required. Additionally, depending on the source and processing of the biological material, specific testing methods for mycobacteria and other contaminants should be included. Alternative microbiological methods (e.g. NAT, flow cytometry) should be applied in order to reduce the time to detection and to provide reliable results before application of a preparation, but should be also assessed for their possible use for the detection of conventionally undetectable micro-organisms.

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Section: Mykoplasmenunclassified

Aktuelles Konzept zur mikrobiologischen Sicherheit von zellbasierten Arzneimitteln

Schurig

Karo

Sicker

et al. 2015

Bundesgesundheitsbl.

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“…13,51 Improper handling usually leads to mixing and contamination, and therefore utmost care should to taken by avoiding conversations, coughing, sneezing, spilling of medium on surfaces and accidental touching of pipettes to surfaces. [52][53][54] …”

Section: Elimination and Prevention Of Microbial Contaminationmentioning

confidence: 99%

Microbial and Viral Contamination of Animal and Stem Cell Cultures: Common Contaminants, Detection and Elimination

Mahmood

Ali

2017

JSRT

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The microbial contamination of animal cell culture, including stem cells, such as the blood stem cells, is a common problem of cell culture laboratories. It is important to identify the types of microorganisms commonly contaminating the cell culture and the sources of contaminating microorganisms, as well as see their effects on cells in culture. This mini review provides a short account of common microbial contaminations of stem cell and animal cell cultures, their detection and elimination, as well as standard practices to ensure a healthy and sterile cell culture, and a brief account of methods used for identification of contaminating microorganisms in cell culture. spoil a culture medium. Keywords7 Residues from germicides used to disinfect incubators, equipments and labs, and impurities in gases used in CO 2 incubators have also been found to affect cell culture.8 However, the major source of microbial contamination is documented to arise by contact with non-sterile supplies, media and solution, chemicals and equipments, and by the particulate matter and aerosols that fall out during transportation and incubation.9 According to a study, 1960-70, the new born bovine serum obtained from commercial suppliers was found to be a major source of bacterial, particularly the mycoplasma, contamination. 10One of the most common contaminants, and also difficult to detect, that has been reported in literature is Mycoplasma contamination. 11Mycoplasma is an intracellular bacterium that may go unnoticed for many passages. Its presence can change several cell properties such as the growth, metabolism, morphology, and genome structure. In literature, the incidences of Mycoplasma contamination have been reported over a period of time since 1956. 12 The incidence of Mycoplasma contamination was 19%, as compared with fungal (8%) and other bacterial (4%) contaminations. The contamination is reported to spread through airborne particles and non-sterile surfaces mainly. 13 In a study on fibroblasts and keratinocyte cell lines, 32.35% cultures were contaminated, and among these, the mycoplasma contaminated cultures were 17.65%, followed by fungal (8.82%) and bacterial (5.88%) contaminants.14 Aspergillus tops the list of fungal contamination of cell cultures. 15M. orale is reported to be the most common contamination that can spread from human oral cavity, and hence making the lab workers a major source of this contamination.10 Bacterial species (Bacillus sp., Enterococcus sp. and Staphylococcus sp) are similarly some major contaminants of animal cell cultures.9 A list of common microbial species and their major sources that have been reported to contaminate animal/stem cell culture is provided in Table 1 & 2, respectively.

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“…The culture medium for each cell line was prepared according to the recommended instructions and supplemented by 10-20 % fetal bovine serum (Sigma, Deisenhofen, Germany) and growth factors as described previously (Molla Kazemiha et al 2011Kazemiha et al , 2014. In order to detect the mycoplasma contamination after harvesting, each cell line was cultured in an antibiotics-free medium for a week (at least 4-5 days) without exchanging the medium (Phelan 2006(Phelan , 2007 (Barile and McGarrity 1983;Rottem et al 2012). …”

Section: Cell Culturesmentioning

confidence: 99%

“…Mycoplasmas are the smallest living organisms with a diameter of about 300-800 nm. They are assumed to be evolved through genomic reduction and reversible evolution from Lactobacillus, Streptococcus and Clostridium Rottem et al 2012). Mostly, mycoplasma genome is circular double-stranded DNA although some species with linear doublestranded DNA genome have been described.…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2015

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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 Ò 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 Ò 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 Ò , indirect DNA DAPI staining and microbial culture methods, respectively. The analysis of the results of the different methods showed that the realtime 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.

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Contamination of Tissue Cultures by Mycoplasmas

Cited by 22 publications

References 116 publications

Aktuelles Konzept zur mikrobiologischen Sicherheit von zellbasierten Arzneimitteln

Aktuelles Konzept zur mikrobiologischen Sicherheit von zellbasierten Arzneimitteln

Microbial and Viral Contamination of Animal and Stem Cell Cultures: Common Contaminants, Detection and Elimination

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

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