A Guide for Ex Vivo Handling and Storage of Stool Samples Intended for Fecal Microbiota Transplantation

Abstract

Owing to the growing recognition of the gut microbiota as a main partner of human health, we are expecting that the number of indications for fecal microbiota transplantation (FMT) will increase. Thus, there is an urgent need for standardization of the entire process of fecal transplant production. This study provides a complete standardized procedure to prepare and store live and ready-to-use transplants that meet the standard requirements of good practices to applied use in pharmaceutical industry. We show that, if time before transformation to transplants would exceed 24 hours, fresh samples should not be exposed to temperatures above 20 °C, and refrigeration at 4 °C can be a safe solution. Oxygen-free atmosphere was not necessary and simply removing air above collected samples was sufficient to preserve viability. Transplants prepared in maltodextrin-trehalose solutions, stored in a -80 °C standard freezer and then rapidly thawed at 37 °C, retained the best revivification potential as proven by 16S rRNA profiles, metabolomic fingerprints, and flow cytometry assays over a 3-month observation period. Maltodextrin-trehalose containing cryoprotectants were also efficient in preserving viability of lyophilized transplants, either in their crude or purified form, an option that can be attractive for fecal transplant biobanking and oral formulation.

Figure 1

Heatmap of log2(condition/baseline) for genus abundance in raw fecal samples from two individuals (S2 and S3). Conditions are combinations of temperature (37 °C, RC for room temperature, and 4 °C), atmosphere (An and Ae for anaerobiosis and aerobiosis, respectively), and time after stool collection (24, 48 and 72 hours). Baseline is genus abundance within two hours of stool collection.

Figure 2

Heatmap of log2(condition/baseline) for metabolite abundance in the same raw samples as on Fig. 1. Conditions are the same as on Fig. 1. Baseline is metabolite abundance within two hours of stool collection.

Figure 3

Heatmap of log2(condition/baseline) for genus abundance in cultures of a raw sample from S3 (the same as that on Figs 1 and 2). Conditions are the same as on Figs 1 and 2. Baseline is genus abundance in the culture within two hours of stool collection.

Figure 4

Heatmap of log2(condition/baseline) for metabolite abundance in cultures of a raw sample from S3 (the same as that on Fig. 3). Conditions are the same as on Fig. 3. Baseline is metabolite abundance in the culture within two hours of stool collection.

Figure 5

Heatmap of log2(condition/baseline) for genus abundance in cultures of fecal transplants (from three individuals S1, S2 and S3) that have been frozen at -80 °C for two weeks. Conditions are combinations of conditioning atmosphere (An and Ae for anaerobiosis and aerobiosis, respectively), diluent (NaCl, MD or TR), and thawing protocol (5 min at 37 °C in a water bath or overnight at 4 °C). Baseline is genus abundance in the transplants before freezing.

Figure 6

Heatmap of log2(condition/baseline) for metabolite abundance in cultures of fecal transplants (from S2, the same as on Fig. 5) that have been frozen at -80 °C for two weeks. Conditions are the same as on Fig. 5. Baseline is metabolite abundance in the transplants before freezing.

Figure 7

Percent live bacteria in transplants prepared in NaCl (a, four donations from subjects S2, S3, S4 and S5), or in either NaCl, or MD, or TR (b, two different donations from subjects S2 -round dots- and S4 -triangular dots-). Transplants were frozen by fractions in a -80 °C standard freezer, and then rapidly thawed at defined times (24h: 24 hours, 1W: 1 week, 1M: 1 month, 3M: 3 months) for live/dead tests by flow cytometry. Viabilities at 0 hour are those measured before freezing.

Figure 8

Percent live bacteria in fresh, frozen or further freeze-dried purified microbiota in either NaCl, or MD, or TR. Samples are ranked in descending order of viability (live/dead test by flow cytometry). Statistics are detailed in Supplementary Table S1. The black bars are 95% confidence intervals.