Biocenter Oulu & Cancer and Translational Medicine Research Unit, Faculty of Medicine, University of Oulu, Oulu, Finland.
Laboratory of Developmental Biology, Disease Networks Research Unit, Faculty of Biochemistry and Molecular Medicine, University of Oulu and Kvantum Institute, Oulu, Finland.
Methods Mol Biol. 2023;2668:211-226. doi: 10.1007/978-1-0716-3203-1_15.
Commensal microbiota has huge impact on the maintenance of human health, its dysregulation being associated with the development of a plethora of diseases. Release of bacterial extracellular vesicles (BEVs) is a fundamental mechanism of systemic microbiome influence on the host organism. Nevertheless, due to the technical challenges of isolation methods, BEV composition and functions remain poorly characterized. Hereby, we describe the up-to-date protocol for isolation of BEV-enriched samples from human feces. Fecal extracellular vesicles (EVs) are purified through the orthogonal implementation of filtration, size-exclusion chromatography (SEC), and density gradient ultracentrifugation. EVs are first separated from bacteria, flagella, and cell debris by size. In the next steps, BEVs are separated from host-derived EVs by density. The quality of vesicle preparation is estimated via immuno-TEM (transmission electron microscopy) for the presence of vesicle-like structures expressing EV markers and via NTA (nanoparticle tracking analysis) for assaying particle concentration and size. Distribution of EVs of human origin in gradient fractions is estimated using antibodies against human exosomal markers with Western blot and ExoView R100 imaging platform. The enrichment for BEVs in vesicle preparation is estimated by Western blot for the presence of bacterial OMVs (outer membrane vesicles) marker and OmpA (outer membrane protein A). Taken together, our study describes a detailed protocol for EV preparation with enrichment for BEVs from feces with a purity level suitable for bioactivity functional assays.
共生微生物群对维持人类健康有巨大影响，其失调与多种疾病的发展有关。细菌细胞外囊泡 (BEV) 的释放是系统微生物组对宿主生物体产生影响的基本机制。然而，由于分离方法的技术挑战，BEV 的组成和功能仍未得到充分描述。在此，我们描述了从人粪便中分离富含 BEV 的样本的最新方案。粪便细胞外囊泡 (EV) 通过过滤、大小排阻色谱 (SEC) 和密度梯度超速离心的正交实施进行纯化。EV 首先通过大小与细菌、鞭毛和细胞碎片分离。在接下来的步骤中，通过密度将 BEV 与宿主来源的 EV 分离。通过免疫 TEM（透射电子显微镜）评估囊泡制剂的质量，以评估存在表达 EV 标志物的囊泡样结构，通过 NTA（纳米颗粒跟踪分析）评估颗粒浓度和大小。使用针对人外泌体标志物的抗体通过 Western blot 和 ExoView R100 成像平台估计人源性 EV 在梯度级分中的分布。通过 Western blot 评估囊泡制剂中 BEV 的富集程度，以检测细菌 OMVs（外膜囊泡）标志物和 OmpA（外膜蛋白 A）的存在。总之，我们的研究描述了一种从粪便中分离富含 BEV 的 EV 并进行纯化的详细方案，其纯度水平适合生物活性功能测定。
