Assessment of the reproducibility of bacterial membrane vesicle isolation and characterization

<b>Aim:</b> This study aimed to evaluate the reproducibility of the isolation and characterization of feces-derived bacterial membrane vesicles. <b>Methods:</b> Human fecal samples (<i>n</i> = 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. <b>Results:</b> 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) <i>P</i> = 0.356] or bacteria (PERMANOVA <i>P</i> = 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. <b>Conclusion:</b> 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.