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

Sebastian D Burz,Christel Béra-Maillet,Joël Doré,Orlane Patrascu,Olivier David,Karine Le Roux,Florence Levenez,Fernanda Fonseca,Fabienne Béguet-Crespel,Patricia Lepage,Anne-Laure Abraham,Catherine Juste,Hervé M Blottière,Carole Schwintner,Stéphanie Cénard,Audrey Chapron

doi:10.1038/s41598-019-45173-4

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.

Highlights

The human intestinal microbiota ensures several essential functions that support maintenance of health and well-being
Dysbiosis is acknowledged as a common feature of numerous non-infectious diseases of modern societies, which have had a constant increase in incidence since the middle of the past century
As microbiota can be modulated by nutrition, drugs or FMT, beneficial functions dependent on intestinal ecological homeostasis can most likely be promoted or restored[17]

Summary

Introduction

The human intestinal microbiota ensures several essential functions that support maintenance of health and well-being. One of them is the prevention of colonization with allochtonous microbes including pathogens, known as barrier effect or competitive exclusion This is dependent on the maintenance of ecological homeostasis of the gut ecosystem[1]. FMT has gained visibility and renewed interest in the clinical domain[21] over the past decade and it may be relevant for disease-associated microbiota alterations, and clinical practice- or daily-life-induced alterations In this domain, current practice is still essentially empirical, benefiting from half a century of compassionate practice and disparate trials, but without a sound knowledge of key determinants of success, nor ecological impact in the long run. Only minimum guidelines exist for the preparation and conservation of fecal transplants, and they are ‘based on what has been described, but never rigorously tested’[20]

Methods

Results

Conclusion