Cryopreservation of Infant Gut Microbiota with Natural Cryoprotectants

Impact of cryoprotective agents on human gut microbes and in vitro stabilized artificial gut microbiota communities

“…In this context, cryopreservation at −80°C represents the most widely exploited protocol for long‐term bacterial storage as it avoids the mechanical, osmotic and oxidative stress to which bacteria are exposed when freeze‐drying is used as storage processing (Barzegari et al., 2014 ; Bircher, Geirnaert, et al., 2018 ; Bircher, Schwab, et al., 2018 ; Smirnova et al., 2019 ). However, since cryopreservation is also not exempt from causing lethal damage to bacterial cells, this storage method requires an accurate evaluation of the components with cryoprotective properties to be used to protect the bacterial biomass from ice formation (Biclot et al., 2022 ; Bircher, Geirnaert, et al., 2018 ; Bircher, Schwab, et al., 2018 ; Chen, Hu, et al., 2022 ; Chen, Huo, et al., 2022 ; Chen, Li, et al., 2022 ; Smirnova et al., 2019 ). In this context, different cryoprotective buffers were assayed for their ability to preserve the viability of 53 bacterial species representing common bacterial taxa of the human gut microbiota, as already pointed out in other studies that used bacterial species representing common taxa of the human gut microbiota (Table S1 ) (Tramontano et al., 2018 ; Zimmermann et al., 2019 ).…”

“…Furthermore, they allow a wide range of taxonomic combinations to always be available in the laboratory, as well as higher reproducibility of the experiments since artificial gut microbiota are not subject to the variations that, instead, occur over time in the microbial community of a donor (Bircher, Geirnaert, et al., 2018 ; Bircher, Schwab, et al., 2018 ; Silverman et al., 2018 ). However, the long‐term storage of the artificial gut microbiota is an essential prerequisite to ensure the viability of the incorporated species and, therefore, to maintain its desired and predetermined bacterial biodiversity (Biclot et al., 2022 ; Bircher, Geirnaert, et al., 2018 ; Bircher, Schwab, et al., 2018 ; Chen, Hu, et al., 2022 ; Chen, Huo, et al., 2022 ; Chen, Li, et al., 2022 ; Smirnova et al., 2019 ).…”

“…However, ice formation may cause lethal damage to bacterial cells, making the use of cryoprotective agents necessary (Bircher, Geirnaert, et al., 2018 ; Bircher, Schwab, et al., 2018 ; Smirnova et al., 2019 ). In this context, the effects of various cryoprotectants on bacterial cell viability and physiology during freezing, storage and thawing have been reported for pure cultures or faecal samples (Biclot et al., 2022 ; Bircher, Geirnaert, et al., 2018 ; Bircher, Schwab, et al., 2018 ; Chen, Hu, et al., 2022 ; Chen, Huo, et al., 2022 ; Chen, Li, et al., 2022 ; Fouhy et al., 2015 ; Li et al., 2023 ). However, there is still little knowledge regarding the type of protective matrices to be used to prevent cryoinjuries for various bacterial species that represent (typical) components of the human gut microbiota or for a mix of bacterial species forming an artificial gut microbiota that lacks the protective matrix naturally present in faecal samples.…”

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

“…The use of two equally effective cryoprotective agents in combination significantly increases the likelihood of successful cryopreservation of the microbial community as a whole. To further enhance the cryoprotective potential of the studied composition, non-penetrating cryoprotectors such as high molecular weight compounds (PEG, PVA) [ 9 ] or trehalose could be considered [ 8 ]. Since our work has limitations related to the use of pure cultures, we plan to create artificial mixes of different bacterial species in future experiments that will better reflect the application of cryoprotective agents to them.…”

“…The most common cryopreservation regimens involved rapid freezing in liquid nitrogen (− 196 °C) which can be followed by transfer to an ultralow temperature freezer (− 80 °C), or slow freezing by direct transfer to a − 80 °C freezer. A few recent studies also focused on using single main cryoprotectants such as 30% glycerol [ 7 ], 10–20% trehalose, betaine, and proline, as well as PBS-based solutions [ 8 ], polymers (PEG, PVA) [ 9 ]. While these methods have shown some success, we believe that utilizing multicomponent media for freezing is a more promising approach to increase the efficiency of cryopreservation.…”