Scientific Publications by FDA Staff

Appl Environ Microbiol 2008 Sep;74(17):5383-91

Biological Enrichment of Mycoplasma Agents using Co-Cultivation with Permissive Cell Cultures.

Volokhov DV, Kong H, George J, Anderson C, Chizhikov VE

Volokhov, DV, US FDA, Ctr Biol Evaluat & Res, Off Vaccine Res & Review, Div Viral Prod,Lab Methods Dev, HFM 470,1401 Rockville Pike, Rockville, MD 20852 USA US FDA, Ctr Biol Evaluat & Res, Off Vaccine Res & Review, Div Viral Prod,Lab Methods Dev, Rockville, MD 20852 USA

Herein, we describe our results on the evaluation 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 (NAT) or biochemical technologies (e.g. MycoAlert Mycoplasma Detection). Of ten 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 (M. 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 reliably detected after 7-day enrichment in MDCK cell culture at 0.05-0.25 cfu/ml contamination levels. 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 the insect cell culture was demonstrated to be temperature dependent, and most efficient growth was observed when incubation temperature was increased from 28 degrees C to 35-37 degrees C. We believe that this type of mycoplasma enrichment is one of most promising approaches for improving the purity and safety testing of cell substrates and other cell derived biologics and pharmaceuticals.