biostudies-literatureUnknown6(4)Verbunt J<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 Extracellular vesicles and circulating nucleic acids728-741https://www.ebi.ac.uk/biostudies/studies/S-EPMC12809393biostudies-literatureAssessment of the reproducibility of bacterial membrane vesicle isolation and characterization.PMC12809393Blaak EECanfora EBarnett DStassen FSavelkoul PJocken JVerbunt JfalseAssessment 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 2025-01-01T00:00:00Z20252026-06-06T14:40:11.878Z2026-05-31T03:10:15.639ZS-EPMC128093934155159810.20517/evcna.2025.71