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Applied and Environmental Microbiology, September 2008, p. 5383-5391, Vol. 74, No. 17
0099-2240/08/$08.00+0     doi:10.1128/AEM.00720-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Biological Enrichment of Mycoplasma Agents by Cocultivation with Permissive Cell Cultures{triangledown}

Dmitriy V. Volokhov,*{dagger} Hyesuk Kong,{dagger} Joseph George, Christine Anderson, and Vladimir E. Chizhikov

Center for Biologics Evaluation and Research, Food and Drug Administration, 1401 Rockville Pike, HFM-470, Rockville, Maryland 20852

Received 27 March 2008/ Accepted 27 June 2008

In this study, we describe our results on the evaluation of the ability of different permissive mammalian cell lines to support the biological enrichment of mycoplasma species known to be bacterial contaminants of cell substrates. The study showed that this approach is able to significantly improve the efficiency of mycoplasma detection based on nucleic acid testing or biochemical technologies (e.g., MycoAlert mycoplasma detection). Of 10 different cell lines (Vero, MDBK, HEK-293, Hep-G2, CV-1, EBTr, WI-38, R9ab, MDCK, and High Five) used in the study, only MDCK cell culture was found to support the efficient growth of all the tested mycoplasmas (Mycoplasma arginini, M. bovis, M. fermentans, M. gallinaceum, M. gallisepticum, M. synoviae, M. hominis, M. hyorhinis, M. orale, M. salivarium, and Acholeplasma laidlawii) known to be most frequently associated with contamination of cell substrates and cell lines in research laboratories or manufacturing facilities. The infection of MDCK cells with serial dilutions of each mycoplasma species demonstrated that these common cell line contaminants can be detected reliably after 7-day enrichment in MDCK cell culture at contamination levels of 0.05 to 0.25 CFU/ml. The High Five insect cell line was also found to be able to support the efficient growth of most mycoplasma species tested, except for M. hyorhinis strain DBS1050. However, mycoplasma growth in insect cell culture was demonstrated to be temperature dependent, and the most efficient growth was observed when the incubation temperature was increased from 28°C to between 35 and 37°C. We believe that this type of mycoplasma enrichment is one of the most promising approaches for improving the purity and safety testing of cell substrates and other cell-derived biologics and pharmaceuticals.

* Corresponding author. Mailing address: U.S. Food and Drug Administration, Center for Biologics Evaluation and Research, Office of Vaccine Research and Review, Division of Viral Products, Laboratory of Methods Development, HFM-470, 1401 Rockville Pike, Rockville, MD 20852. Phone: (301) 827-8757. Fax: (301) 827-9531. E-mail: dmitriy.volokhov{at}fda.hhs.gov

{triangledown} Published ahead of print on 7 July 2008.

{dagger} These authors contributed equally to this work.

Applied and Environmental Microbiology, September 2008, p. 5383-5391, Vol. 74, No. 17
0099-2240/08/$08.00+0     doi:10.1128/AEM.00720-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.





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