Mycoplasma testing of cell substrates and biologics: Review of alternative non-microbiological techniques

Volokhov, Dmitriy V.; Graham, Laurie; Brorson, Kurt; Chizhikov, Vladimir E.

doi:10.1016/j.mcp.2011.01.002

Cited by 63 publications

(61 citation statements)

References 62 publications

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“…In this study, the false negative results for enzymatic method were observed in only two cell lines i.e., PEER (NCBI C511) and SH-SY5Y (NCBI C611). In the other 28 cases, the mycoalert Ò results were concordant with the real-time PCR results (Mariotti et al 2008;Markoullis et al 2009;Volokhov et al 2011;Molla Kazemiha et al 2014).…”

Section: Discussionsupporting

confidence: 59%

“…Direct microbial culture, as the standard reference technique, is frequently applied as a sensitive and reliable method for the detection of mycoplasma in cell cultures. Slow rate of mycoplasma growth and difficulties in the interpretation of the results are considered as the disadvantages of microbial cultivation (Volokhov et al 2011). In our study, there were false negative results for 8 cases (26.66 %) using this method.…”

Section: Discussionmentioning

confidence: 99%

“…Thermal cycle setting for StepOne Plus TM real-time PCR System was as follows: initial polymerase activation at 95°C for 2 min and 45 cycles of denaturation at 95°C for 30 s, annealing at 55°C for 30 s and extension at 60°C for 45 s. Mycoplasma contaminations were analyzed by the assessment of the increase in fluorescence signal during the real-time PCR test in FAM channel. The inhibition internal control amplification was indicated in VIC channel (Espy et al 2006;Volokhov et al 2011). …”

Section: Real-time Pcr For the Detection Of Mycoplasma Contamination mentioning

confidence: 99%

“…In this study, direct microbial culture (as a gold standard), enzymatic mycoalert Ò mycoplasma detection kit, indirect DNA DAPI staining, real-time PCR and PCR methods were utilized for the detection of mycoplasma contamination in the cell collections of the National Cell Bank of Iran. The sensitivity, specificity, accuracy, and predictive value of positive and negative results of these methods were compared in order to find the best method Razin 1994;Harasawa et al 2005;Volokhov et al 2011;Uphoff and Drexler 2014).…”

Section: Introductionmentioning

confidence: 99%

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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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Section: Discussionsupporting

confidence: 59%

Section: Discussionmentioning

confidence: 99%

Section: Real-time Pcr For the Detection Of Mycoplasma Contamination mentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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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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“…The plate was carefully sealed to prevent contamination and evaporation and incubated at 37°C for 4 weeks. The formation of egg form or non-typical colonies of mycoplasm was investigated by light microscopy every 3 days (Dabrazhynetskaya et al 2011;Volokhov et al 2011;Peredeltchouk et al 2011). …”

Section: Microbial Culture For Detection Of Mycoplasma Contamination mentioning

confidence: 99%

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

et al. 2014

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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 Ò and molecular. Enzymatic evaluation was performed using the mycoalert Ò 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 Ò 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).

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Pre‐Plated Cell Lines for ADMETox Applications in the Pharmaceutical Industry

Torres¹,

Sáfár

Vázquez‐Sánchez

et al. 2015

CP Toxicology

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Membrane transporters significantly modulate membrane permeability of endobiotics and xenobiotics, such as bile acids and drugs, respectively. Various in vitro methods have been established for both ATP-binding cassette (ABC) transporters to examine cellular efflux and uptake, and for solute carriers (SLC) to examine cellular uptake of substrates. Cell-based systems are the models of choice to test drug-transporter interactions as well as drug-drug interactions for research and regulatory purposes, albeit, for low passive permeability substrates of ABC transporters, vesicular uptake assays are also recommended. Commercially available pre-plated cells (e.g., immortalized or transfected) offer a useful alternative to in-house cell culture. Three main methods are known to manufacture pre-plated cultures: regular culture medium with vacuum seal, cryopreserved delivery, and the solid shipping media technology. The regular culture medium and the solid shipping media technologies provide ready-to-use models for end users. Models expressing a broad selection of transporters are available in pre-plated formats for absorption, distribution, metabolism, excretion, and toxicity (ADMETox) studies. Conversely, the application and utility of pre-plated cultures coupled with personal experiences have not been extensively covered in published research papers or reviews, despite availability and significant use of pre-plated products in the pharmaceutical industry. In this overview, we will briefly describe: 1) in vitro tools commonly used for ADMETox testing; 2) methods employed in manufacturing, shipment and preparation of pre-plated cell lines; 3) cell-membrane barrier models currently available in pre-plated format to reproduce passage restriction of physiological barriers to certain compounds; and 4) recommended pre-plated cell lines overexpressing uptake transporters for ADMETox applications.

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Mycoplasma testing of cell substrates and biologics: Review of alternative non-microbiological techniques

Cited by 63 publications

References 62 publications

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

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

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

Pre‐Plated Cell Lines for ADMETox Applications in the Pharmaceutical Industry

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