A Guide for Ex Vivo Handling and Storage of Stool Samples Intended for Fecal Microbiota Transplantation
Authors: Joël Doré
Catherine Juste
Carole Schwintner
Fernanda Fonseca
Karine Le Roux
Audrey Chapron
Sebastian D Burz
Fabienne Béguet-Crespel
Stéphanie Cénard
Patricia Lepage
Hervé M Blottière
Florence Levenez
Anne-Laure Abraham
Orlane Patrascu
Christel Béra-Maillet
Olivier David
Journal: Scientific Reports (2019)
Discipline: Multidisciplinary
Keywords: gut microbiota; fecal microbiota transplantation (FMT); standardization; transplants; production
Affiliations: Micalis Institute, INRA, AgroParisTech, Université Paris-Saclay, 78350, Jouy-en-Josas, France., Université Paris-Saclay, MetaGenoPolis, INRA, 78350, Jouy-en-Josas, France., MaIAGE, INRA, Université Paris-Saclay, 78350, Jouy-en-Josas, France., GMPA, INRA, AgroParisTech, Université Paris-Saclay, 78850, Thiverval-Grignon, France., MaaT Pharma, Pharmaceutical Development, 69007, Lyon, France., Micalis Institute, INRA, AgroParisTech, Université Paris-Saclay, 78350, Jouy-en-Josas, France. .

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. 

Retracted: No

PubPeer comments:
 The listed ratios are not clear to me. Two maltodextrin-trehalose cocktail recipes in the weight ratios of 3:1 or 1:3 in normal saline were tested and compared to normal saline. They are referred as to u2018MDu2019, u2018TRu2019 and u2018NaClu2019. Maltodextrin and trehalose were dissolved in normal saline (9.0u2009g L NaCl) at 40u2009u00b0C, to respective final concentrations of 18.75% and 6.25% (w v) for MD, or 6.25% and 18.75% (w v) for TR. Does this mean 3 parts maltodextrin to 1 part saline? Or 3 parts maltodextrin to 1 part stool? What does  3:1 OR 1:3 mean? It suggests you tested MD at a 3:1 ratio and a 1:3 ratio but I don t see any comparison of that. The final concentrations percentages seem to suggest it was 1 part maltodextrin to 3 parts saline, or to 3 parts stool. But that s not clear either. If it s 3 parts MD to 1 part saline that seems like it would be quite firm and thus difficult to mix in with stool that is type 2 or 3 on the Bristol Stool Scale.  maltodextrin-trehalose cocktail seems to imply that you mixed maltodextrin and trehalose together into one cocktail, but looking at the figures images it seems you did not mix them. Perhaps  maltodextrin-trehalose cocktail should be changed to  maltodextrin or trehalose cocktail . , 
 receipe#1: 18.75 g maltodextrin + 6.25 g trehalose 100mL final solution (solvant NaCl 9g L) receipe#2: 6.25 g maltodextrin + 18.75 g trehalose 100mL final solution (solvant NaCl 9g L) , 
 Oh ok. Thank you for clarifying. Did I miss at what concentration the MD-trehalose solutions were mixed in with the stool, or is that not in the paper? IE: stool estimated to be 80% of final mix and the MD-trehalose solution 20%. It seems there s only the  final concentration percentages, which are percentages of the MD-TR in saline, but no  final concentration percentage that includes the stool? Nevermind, I found it:  Four ml of diluent were added per g of stool . That s approximately a 1:1 ratio. , 
 Four ml of diluent were added per g of stool . - ratio ~4:1, i.e. 1 g stool in ~5 ml final , 
 Oops. Thanks for the correction. ,