Contamination by mycoplasma

Although primary cell cultures are usually free from contamination by mycoplasmas, many cell strains and lines in use are contaminated. This probably arose from the use of animal sera contaminated with A. laidlawii and M. arginini or pig trypsin contaminated with M. hyorhinis. These are probably no longer a significant source of contamination and, although M. hominis, M. pharyngis and M. salivarium are readily isolated from human mouths and throats, the most frequent source of contamination today is from working with contaminated cells. It has been estimated that 50-95% of cells in use today are contaminated with mycoplasma. 

Arindam Bose (Pfizer Central Research) further discussed the ICH documents and presented a rationale for the recommended combination of test procedures and process clearance validations required to demonstrate that marketed biopharmaceuticals are free of adventitious agents. He showed that testing of Pre-Seed Stock (PSS), the Master Cell Bank (MCB), and the Working Cell Bank (WCB) is required to demonstrate that they are free from contamination by mycoplasma, bacteria, molds, and yeasts. In addition, viral clearance validation studies must be performed on scaled down versions of each chromatographic step and the viral inactivation/removal step employed in the product purification scheme. Finally, clearance studies must be conducted with a panel of relevant and model viruses (typically three to four) to establish that the purification scheme will indeed purge any viruses that may be inadvertently introduced during processing. 

While bacterial and fungal contaminations represent an added concern, in most instances they are overt and easily detected, and therefore have less serious consequences than the more insidious contaminations by mycoplasma. That the presence of these microorganisms in cultured cell lines often negates research findings entirely has been stated repeatedly over the years (Barile et al., 1973 Hay et al., 1989). However, the difficulties of detection and prevalence of contaminated cultures in the research community suggest that the problem cannot be overemphasized. These and related difficulties associated with use of cell lines obtained from different sources can be avoided if one acquires stocks from a centralized cell banking agency which applies appropriate quality control (Hay, 1992). 

The rhizomes of Alpinia qfficinarum were purchased from Nuherbs Co. (Oakland, CA). Du-145 prostate tumor cells were obtained from the American Type Culture Collection (ATCC). Cells were maintained at 37°C in an atmosphere of 5% CO2 and grown in Roswell Park Memorial Institute (RPMI) media 1640 (GIBCO/BRL) with 10% fetal bovine serum and with 5% penicillin and streptomycin. Cells ere routinely checked and foimd to be free of contamination by mycoplasma. 

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See also in sourсe #XX -- [ ]

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