Method of preparing a faecal microbiota sample
The present invention relates to a method for preparing a fecal 5 microbiota sample of a donor subject. Said method includes the following steps: a) collecting at least one fecal microbiota sample from the donor subject; b) within a period of less than 5 minutes after collecting the sample, placing said sample obtained in step a) in an oxygen-tight collection device; c) mixing the sample obtained in step b) with at least one aqueous saline 10 solution containing at least one cryoprotectant and/or a filling agent; d) optionally, filtering the mixture obtained in step c), in particular by means of a filter comprising pores having a diameter of less than or equal to 0.7 mm, preferably less than or equal to 0.5 mm; and e) storing the mixture obtained in step c) or d) by freezing said mixture at a temperature 15 of between −15° C. and −100° C. Steps b) to e) are carried out in anaerobiosis.
1. A method of preparing a sample of faecal microbiota from a donor subject, comprising the following steps:
a) taking at least one sample of faecal microbiota from the donor subject,
b) within 5 minutes following taking of the sample, placing said sample obtained in a) in an oxygen-tight collecting device,
c) mixing the sample obtained in b) with at least one saline aqueous solution comprising 5-1.5% w/v of at least one cryoprotectant selected from the group consisting of trehalose, galactose-lactose and combinations thereof; and 5-15% w/v of at least one maltodextrin to form a mixture,
d) optionally, filtering the mixture obtained in c), and
e) storing the mixture obtained in c) or d) by freezing at a temperature between −1.5° C. and −100° C.,
wherein steps b) to e) are carried out under anaerobiosis and wherein bacterial functional integrity of the faecal microbiota is preserved, and
wherein the at least one saline aqueous solution further comprises sodium ascorbate and cysteine hydrochloride monohydrate.
2. The method of claim 1 , wherein the sample placed in the collecting device of step b) undergoes a transportation step prior to step c).
3. The method of claim 1 , wherein the sample placed in the collecting device of step b) is incubated at a temperature comprised between 33° C. and 40° C. for a maximum time of 75 h, between steps b) and c).
4. The method of claim 1 , wherein the saline aqueous solution comprises:
at least one salt chosen from sodium chloride, calcium chloride, magnesium chloride, potassium chloride, sodium gluconate and sodium acetate.
5. The method of claim 1 , wherein the at least one maltodextrin is a mixture of maltodextrins.
6. The method of claim 1 , further comprising a step f) of thawing the frozen sample obtained in e), under anaerobiosis, up to ambient temperature.
7. A method for transplantation of autologous or allogenic faecal Microbiota , comprising introducing by transplantation into a subject the thawed sample of donor fecal Microbiota prepared according to the method of claim 6 .
8. A method for treating intestinal dysbioses, comprising introducing by transplantation into a patient in need thereof the thawed sample of donor fecal Microbiota prepared according to the method of claim 6 .
9. The method of claim 1 , wherein the filter used in step d) comprises pores of diameter less than or equal to 0.7 mm.
10. The method of claim 1 , wherein the filter used in step d) comprises pores of diameter less than or equal to 0.5 mm.
11. The method of claim 1 , wherein freezing in step e) is at a temperature between −60° C. and −90° C.
12. The method of claim 1 ; where the at least one saline aqueous solution comprises 0.5% w/v sodium L-ascorbate and 0.1% w/v cysteine hydrochloride monohydrate.
13. The method of claim 1 , wherein the amount of cryoprotectant and maltodextrin in the at least one saline aqueous solution is 20% w/v.
14. The method of claim 13 , wherein the amount of cryoprotectant is 15% w/v trehalose, galactose-lactose or a combination thereof and the amount of maltodextrin is 5% w/v.
15. The method of claim 13 , wherein the amount of cryoprotectant is 5% w/v trehalose, galactose-lactose or a combination thereof and the amount of maltodextrin is 15% w/v.