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Assessment of the reproducibility of bacterial membrane vesicle isolation and characterization

Jari Verbunt,J. Jocken,E. Canfora,David J. M. Barnett,E. Blaak,P. Savelkoul,Frank R. M. Stassen

Published 2025 in Extracellular Vesicles and Circulating Nucleic Acids

ABSTRACT

Aim: This study aimed to evaluate the reproducibility of the isolation and characterization of feces-derived bacterial membrane vesicles. Methods: Human fecal samples (n = 12) stored at -80 °C were thawed, sampled, and then refrozen. From these samples, bacterial membrane vesicles were isolated through ultrafiltration, ultracentrifugation and size exclusion chromatography. Vesicle-associated DNA was characterized by marker [16 ribosomal DNA (rDNA)] sequencing to determine composition. The same fecal samples were thawed again after > 6 months of storage at -80 °C to repeat this procedure. Compositions and other vesicle characteristics were compared to investigate effects of storage and freeze/thawing on sample stability. In addition, for four of the fecal aliquots, the bacteria were subjected to marker gene sequencing alongside their derived membrane vesicles. Results: No significant differences were observed in the pre- and post freeze/thawing composition of feces-derived bacterial membrane vesicles [permutational multivariate analysis of variance (PERMANOVA) P = 0.356] or bacteria (PERMANOVA P = 0.721) as determined by 16S rDNA sequencing. Additionally, no significant differences were observed in vesicle size, concentration, and associated protein or DNA content. These results indicate that, long-term storage of feces at -80 °C and an additional freeze/thawing cycle does not induce compositional or qualitative changes to vesicle repertoires. Conclusion: These reproducibility findings hold great relevance for research on (gut)bacteria derived membrane vesicles. Our results indicate that fecal samples can be stably preserved at -80 °C for bacterial and vesicle isolations as their characteristics remain stable over time.

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