Cryopreservation of Infant Gut Microbiota with Natural Cryoprotectants

A growing body of evidence has demonstrated the importance of the gut microbiome in human health. In general, fecal microbial samples are used to study the mechanisms of relevant diseases. In this context, it is worth mentioning that an optimized cryopreservation method is urgently needed to successfully perform clinical diagnosis, therapy, and scientific investigations of the gut microbiome without affecting its viability and biological activity. In this study, we aimed to test the relative cryopreservation efficiency of different nontoxic natural cryoprotectants using infant fecal and meconium samples. First, we selected two facultative and two obligate anaerobic bacteria as the experimental gut microbial strain to compare these cryoprotectants' toxicity and concentration-dependent bacteria viabilities after cryopreservation, then the viabilities and bacterial diversity of mixed facultative and obligate anaerobic bacteria. Finally, we explored the effects of optimized cryoprotectants for meconium and infant feces after cryopreservation using 16S rRNA sequencing analyses. In addition, to better understand the effectiveness of these cryoprotectants, we used different freeze-thaw conditions mimicking real-life situations in the process of distribution. We found that the better choice for the infant fecal sample's cryopreservation was 100 mg/mL trehalose, whereas 200 mg/mL trehalose/betaine was the optimum choice for meconium cryopreservation. We hope that our results will shed light on the importance of natural cryoprotectants toward the long-term and stable viability of invaluable human gut microbial specimens.