Abstract
OBJECTIVE: The need for innovative techniques to preserve microbiota for extended periods, while maintaining the species composition and quantitative balance of the bacterial community, is becoming increasingly important. To address this need, we propose an efficient approach to cryopreserve human gut microbiota using a two-component cryoprotective composition comprising fetal bovine serum (FBS) and 5% dimethyl sulfoxide (DMSO). Fetal serum is a commonly utilized component in the freezing media for eukaryotic cells, however, its effects on prokaryotic cells have not been extensively researched. RESULTS: In our study, we demonstrated the high efficiency of using a two-component cryoprotective medium, FBS + 5% DMSO, for cryopreservation of human gut microbiota using three different methods. According to the obtained results, the intact donor microbiota was preserved at a level of 85 ± 4% of the initial composition based on fluorescent analysis using the LIVE/DEAD test. No differences in survival were observed when comparing with pure DMSO and FBS media. The photometric measurement method for growth of aerobic bacteria (A. johnsoni), facultative anaerobes (E. coli, E. faecalis), microaerophilic (L. plantarum), and obligate anaerobic bacterial cultures (E. barkeri, B. breve) also demonstrated high viability rates of 94-98% in the two-component protective medium, reaching intact control levels. However, for anaerobic microflora representatives, serum proved to be a more suitable cryoprotectant. Also, we demonstrated that using cryoprotective media with 50-75% FBS content is enough to preserve a significant level of bacterial cell viability, from an economic standpoint.