Human gut microbiota preservation for functional studies: in-vitro comparison of common cryoprotective agents. uncultured bacterium

The protocol to test the preservation capabilities was based on a standardized procedure using the ECSIM, a chemostat-based fermentation system establishes to maintain microbiota. The initial fresh stool sample (1 g) was first cultivated in 5 mL, then 100 mL and then 1 L of a standard artificial gut medium and then either immediately tested in the P-ECSIM or preserved for 3 and 6 months with cryoprotectants before being next used for inoculation of the P-ECSIM. Three intracellular protecting agents were tested alone and in combinations (i) 10% (v/v) glycerol, the most current one, (ii) 10% (v/v) dimethyl sulfoxide (DMSO), which possesses a much greater penetrating power and (iii) 10% (w/v) polyethylene glycol-4000 (PEG-4000), an emulsifying agent with high molecular weight. Overall design: Three-condition experiment (0 hour,3 months and 6 months), Control versus 14 different preservation conditions, Biological replicates:1