Detection of microbial contaminants in mammalian cell cultures using a new fluorescence-based staining method: Microcolonies detection using fluorescence

Abstract

Aims Microbial contamination of cell culture production processes is a current concern for biopharmaceutical industries. Traditional testing methods require several days to detect contamination and may advantageously be replaced by a rapid detection method. We developed a new method combining membrane filtration to microcolonies fluorescence staining method (MFSM) and compared it to epifluorescence microscopy. Methods and results Both methods were used to detect bacteria in CHO cells cultures. The epifluorescence microscopy showed to be limited by filterability, media interference and nonrobustness issues, whereas MFSM enabled consistent detection of Bacillus cereus, Staphylococcus epidermidis and Propionibacterium acnes after, respectively, 8, 9 and 48 h of incubation. Thanks to the nondestructive feature of the MFSM, stained membranes could be reincubated on culture media to yield visible colonies used for identification. Conclusions The new method described in this study showed its ability to detect microbial contaminants in cell culture samples with time-to-results from 2-5 times shorter than the traditional testing method. Significance and impact of the study The MFSM can be used as monitoring tool for cell cultures to significantly shorten detection times of microbial contamination, while preserving the ability to identify the contaminants and their viability.