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Updated: Sep 4, 2025

Therapeutic Evaluation of Fecal Microbiota Transplantation in an Interleukin 10-Deficient Mouse Model
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Fresh Versus Frozen Stool for Fecal Microbiota Transplantation-Assessment by Multimethod Approach Combining

Jaroslaw Bilinski1, Mikolaj Dziurzynski2, Pawel Grzesiowski3

  • 1Department of Hematology, Transplantation and Internal Medicine, Medical University of Warsaw, Warsaw, Poland.

Frontiers in Microbiology
|July 18, 2022
PubMed
Summary

Freezing whole stool for fecal microbiota transplantation (FMT) without cryoprotectants significantly reduces bacterial viability and alters community structure. Freshly prepared FMT is superior to frozen preparations for maintaining bacterial quality and diversity.

Keywords:
conservationculturingfecal microbiota transplantationflow cytometrygut microbiotanext-generation sequencingviability

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Area of Science:

  • Microbiology
  • Gastroenterology
  • Biotechnology

Background:

  • Fecal microbiota transplantation (FMT) is a promising therapeutic strategy.
  • Standardizing FMT preparation is crucial for treatment efficacy.
  • The impact of cryopreservation on fecal samples for FMT is not fully understood.

Purpose of the Study:

  • To compare the quality of FMT preparations from fresh feces versus feces frozen at -30°C without cryoprotectants.
  • To test the hypothesis that frozen whole stool is equipotent to fresh FMT preparation.
  • To evaluate the effects of freezing on bacterial viability, cultivability, and community structure.

Main Methods:

  • Fecal samples were processed fresh or after freezing at -30°C.
  • Bacterial viability was assessed using flow cytometry with cell staining.
  • Culturable bacterial communities were analyzed using aerobic and anaerobic culturing.
  • Community structure was determined by next-generation sequencing (NGS) with weighted UniFrac PCoA.

Main Results:

  • Flow cytometry revealed a significant decrease in viable cells (70% to 15%) and an increase in dead/unknown cells after freezing.
  • Freezing drastically altered the structure of cultivable bacterial communities, with notable reductions in Actinobacteria and Bacilli.
  • NGS analysis showed a clear separation between fresh and frozen samples, attributed to changes in Bacteroidales and Clostridiales abundance.

Conclusions:

  • Whole stool freezing without cryoprotectants significantly impacts bacterial viability, cultivability, and community biodiversity.
  • The tested freezing protocol is not equipotent to fresh FMT preparation.
  • Further investigation is needed to identify the "unknown" cell fraction observed after freezing.