FBS-based cryoprotective compositions for effective cryopreservation of gut microbiota and key intestinal microorganisms

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.

Keywords: Bacterial cultures; Cryopreservation; Fetal bovine serum; Gut microbiota.

Fig. 1

Viability of Escherichia coli after cryopreservation (4 days at − 196 °C) in the media 5% DMSO, 100% FBS, and a combination of 5% DMSO and 95%FBS by three different methods: microtiter plate-based cultivation (A), (B), (n = 3); fluorescent LIVE/DEAD assay (C) (n = 10), conventional microbiological cultivation (D), (n = 10). Abs—optical density (600 nm). Columns marked with the same letter are not significantly different at p < 0.05, according to the Mann–Whitney nonparametric criterion

Fig. 2

Viability of five bacterial cultures (microtiter plate-based cultivation, n = 3) and whole human gut microbiota (LIVE/DEAD assay, n = 15) after cryopreservation (4 days at − 196 °C, 24 h of growth) in the media 5% DMSO, 100% FBS, and a combination of 5% DMSO and 95% FBS. Abs – optical density (600 nm). A Eubacterium barkeri, B Bifidobacterium breve, C Lactobacillus plantarum, D Enterococcus faecalis, E Acinetobacter johnsoni, F whole microbiota. Columns marked with the same letter are not significantly different at p < 0.05, according to the Mann–Whitney nonparametric criterion

Fig. 3

The impact of FBS concentrations on the survival of six bacterial cultures after cryopreservation (4 days at − 196 °C) (n = 3) in pure FBS and FBS + 5% DMSO mixtures. Values are normalized to intact control level (100%). A Eubacterium barkeri BBifidobacterium breve, C Lactobacillus plantarum, D Enterococcus faecalis, E Acinetobacter johnsoni, F Escherichia coli. Points marked with the same letter are not significantly different at p < 0.05, according to the Mann–Whitney nonparametric criterion