Biological Enrichment of Mycoplasma Agents by Cocultivation with Permissive Cell Cultures

Abstract

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). 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.

Detection of Mollicutes in bioreactor samples by real-time transcription-mediated amplification

“…To mimic bioreactor contaminations, we have performed cocultures experiments with adherent and suspension cells. In these co-culture conditions, each species grew differently in CHO cell culture as already observed for different Mycoplasma species in Vero cell culture (Volokhov et al 2008). Mycoplasma gallisepticum and M. arginini have shown an exponential growth all along the culture and were easily detected using our method.…”

“…To mimic bioreactor contaminations, we have performed co‐cultures experiments with adherent and suspension cells. In these co‐culture conditions, each species grew differently in CHO cell culture as already observed for different Mycoplasma species in Vero cell culture (Volokhov et al. 2008).…”

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

“…These specific enzymes provide a rapid screening method for sensitive detection of mycoplasma contamination in cell cultures. The enzymes are released during viable mycoplasma lysis and able to transform adenosine diphosphate (ADP) into adenosine triphosphate (ATP) by reaction with carbamoyl phosphate or acetyl phosphate (Mariotti et al 2008;Volokhov et al 2008). The level of ATPs in each sample was measured before (Reading A) and after (Reading B) the addition of Mycoalert Ò substrate.…”

Molecular Diagnostics of Mycoplasmas: Perspectives from the Microbiology Standpoint

“…Another culture-based enrichment approach for improvement of PCR detection of mycoplasmas is the cocultivation of these bacteria with permissive immortalized mammalian and/or insect cell lines [Kong et al, 2007;Volokhov et al, 2008]. Although this approach has been design for intentional screening of cell-derived biological and pharmaceutical products, including vaccines and cell culture substrates, it is a potential tool for biological enrichment of normally-sterile clinical specimens such as CFS, sera, synovial fluid, etc.…”