Assessment of extracellular vesicle isolation methods from human stool supernatant

Abstract

Extracellular vesicles (EVs) are of growing interest due to their potential diagnostic, disease surveillance, and therapeutic applications. While several studies have evaluated EV isolation methods in various biofluids, there are few if any data on these techniques when applied to stool. The latter is an ideal biospecimen for studying EVs and colorectal cancer (CRC) because the release of tumour markers by luminal exfoliation into stool occurs earlier than vascular invasion. Since EV release is a conserved mechanism, bacteria in stool contribute to the overall EV population. In this study, we assessed five EV separation methods (ultracentrifugation [UC], precipitation [EQ‐O, EQ‐TC], size exclusion chromatography [SEC], and ultrafiltration [UF]) for total recovery, reproducibility, purity, RNA composition, and protein expression in stool supernatant. CD63, TSG101, and ompA proteins were present in EV fractions from all methods except UC. Human (18s) and bacterial (16s) rRNA was detected in stool EV preparations. Enzymatic treatment prior to extraction is necessary to avoid non‐vesicular RNA contamination. Ultrafiltration had the highest recovery, RNA, and protein yield. After assessing purity further, SEC was the isolation method of choice. These findings serve as the groundwork for future studies that use high throughput omics technologies to investigate the potential of stool‐derived EVs as a source for novel biomarkers for early CRC detection.

Identification of faecal extracellular vesicles as novel biomarkers for the non‐invasive diagnosis and prognosis of colorectal cancer

“…Gram‐positive and negative bacteria are essential members of the intestinal environment, which release bacterial extracellular vesicles (BEVs) through different formation routes (Brown et al., 2015; Schwechheimer & Kuehn, 2015; Toyofuku et al., 2019). Accumulating evidence has proved that fEVs are a collection of BEVs and host‐derived eukaryotic EVs (Northrop‐Albrecht et al., 2022; Tulkens et al., 2020). At present, the separation of subpopulations in fEVs is still dependent on ultrafiltration (UF) followed by density gradient centrifugation and size‐exclusion chromatography (SEC), making it a labour‐intensive and time‐consuming procedure (Kameli et al., 2021; Tulkens et al., 2020).…”

Integrating Transcriptomics and Metabolomics to Explore the Novel Pathway of Fusobacterium nucleatum Invading Colon Cancer Cells

“…In the tumor microenvironment, extracellular vesicles of microbial origin play an important role in disease diagnosis, monitoring, and treatment [ 57 ]. Fusobacterium nucleatum ( Fn ) plays an important role in the development of colon cancer as a conditionally pathogenic bacterium.…”

Bacterial extracellular vesicles: A position paper by the microbial vesicles task force of the Chinese society for extracellular vesicles

“…162 After transportation to the laboratory, the stool can be aliquoted or dissolved in solution, and then frozen at −80 °C. 163 If you do the latter, selecting the sterile buffer (e.g., phosphate buffer saline) to dissolve the stool is key in preprocessing. The dilution ratio is at least w/v (g/ mL) = 1/10 to prevent oversaturation.…”

“…Sampling kits could be evaluated by combining the subject preference and previous or pre‐experimental BEV analysis results 162 . After transportation to the laboratory, the stool can be aliquoted or dissolved in solution, and then frozen at −80 °C 163 . If you do the latter, selecting the sterile buffer (e.g., phosphate buffer saline) to dissolve the stool is key in pre‐processing.…”