Name: Establishment of a Density Gradient Centrifugation System to Isolate and Purify Bacterial Extracellular Vesicles (BEVs) from Human Faeces
Uploaded: 2023-09-01T14:30:00
Description: Watch this Scientific Journal Video about Establishment of a Density Gradient Centrifugation System to Isolate and Purify Bacterial Extracellular Vesicles BEVs from Human Faeces at JoVE.com

establishment of a density gradient centrifugation system to isolate and purify bacterial extracellular vesicles (bevs) from human faeces

用于微生物群多组学的增强细菌细胞外囊泡分离

The gut is widely recognized as the organ harboring the most abundant microbial communities in the human body, with over 90% of bacteria involved in colonization and multiplication1,2. Extensive evidence has demonstrated that the gut microbiota modulates the gut microenvironment and simultaneously interacts with dysfunction in distant organs, primarily through an impaired intestinal barrier3,4. Mounting evidence indicates a correlation between the imbalance of gut microbiota and the progression of inflammatory bowel disease (IBD)5,6, as well as cognitive disorders through the gut-brain axis5,6,7,8. Bacterial extracellular vesicles (BEVs) produced by bacteria play significant roles in these pathological processes.
BEVs are nanoscale particles encapsulating bacterial derivatives, with diameters ranging from 20 to 400 nm. They have been demonstrated to facilitate interactions between bacteria and their host organisms9,10. Despite their invisibility, these particles have garnered increasing attention from researchers due to their prospective broad applications as diagnostic biomarkers, therapeutic targets, and drug delivery vehicles11. Human feces, often used as biospecimens for studying BEVs, predominantly sourced from gut bacteria, contain a complex mixture of water, bacteria, lipids, proteins, undigested food residue, and exfoliated epithelial cells among others. The intricate fecal composition poses challenges to the isolation and purity of BEVs, thereby impeding a comprehensive, objective, and realistic analysis of BEVs. Hence, effective strategies to minimize interference from contaminating components and enhance the yield of BEVs have emerged as critical issues warranting immediate attention.
Existing isolation strategies largely rely on techniques such as ultra-high-speed centrifugation (UC), density gradient centrifugation (DGC), and size exclusion chromatography (SEC)12,13,14,15,16,17. Currently, DGC is one of the most widely applied methods in the field of BEV separation, encompassing two sedimentation-floating modes, &#34;Top-down&#34; and &#34;Bottom-up&#34;, which are determined by the initial loading position of the sample. These methodologies differentiate extracellular vesicles (EVs) from other components based on size and density disparities, yielding variable purity and recovery rates. Prior research has indicated that single-approach strategies are insufficient for adequately separating EVs from soluble proteins in body fluid samples, such as lipoprotein in blood18 and Tamm-Horsfall protein in urine19. Additionally, the size distribution of eukaryotic extracellular vesicles (EEVs) often overlaps with that of BEVs, thereby necessitating further methodological enhancements to optimize BEV yield. Consequently, advancing the study of BEVs hinges on the development of effective separation and purification methodologies. Notably, Tulkens et al15 employed an orthogonal biophysical strategy to separate fecal BEVs from EEVs, in which the centrifugation time of a Bottom-up DGC mode was up to 18 h. In contrast, this study reduced it to 7 h, greatly saving the gradient-ultracentrifugation time and simplifying the process.
In the present study, we isolated and purified fecal BEVs employing two DGC modes under optimized buffer conditions, after enriching BEVs with a range of differential centrifugation speeds, from low to extremely high velocity. Evaluations based on morphology, particle size, and concentration indicated a commendable performance by this enhanced method. This study could serve as the foundation for future research, extending its applications to a broader domain, and offering insights into the heterogeneity of BEVs within the human body. It also contributes to the standardization of BEV separation and analysis techniques.

作者聚焦：加速细菌细胞外囊泡分离和异质性的研究 

使用密度梯度离心从人粪便中分离和纯化细菌胞外囊泡

Our study isolates, purifies, and characterize bacterial extracellular vesicles from physicists using density gradient centrifugation. We aim to address the separation of BEVs from contaminants and provide a reliable method for researchers. Our protocol reduces centrifugation time from 18 to 7 hours.Top down mode improves BEV separation and reduces contamination from liver proteins. Our method simplifies the separation of BEVs, setting a standard for future studies. It can be applied to other body fluids with appropriate modification, enhancing our understanding and facilitating the study of BEVs revealing their inherent heterogeneity within the human body.

Watch this Scientific Journal Video about Establishment of a Density Gradient Centrifugation System to Isolate and Purify Bacterial Extracellular Vesicles BEVs from Human Faeces at JoVE.com

Establishment of a Density Gradient Centrifugation System to Isolate and Purify Bacterial Extracellular Vesicles (BEVs) from Human Faeces  

建立密度梯度离心系统，从人类粪便中分离纯化细菌胞外囊泡（BEV）

首先，取 500 微升制备的 PBS/BEV 溶液。将其与三毫升 PBS 混合，并使用 1， 000 微升移液管轻轻混合。将 31 毫升超速离心管放在带孔的泡沫板上，并用向下箭头标记管。然后垂直握住1,000微升移液管，并在管底部加入3毫升50%碘克沙醇溶液。将管架放在管开口下方的泡沫板水平上方，并将管子倾斜至 70 度角。使用1， 000微升移液管，在50%碘克沙醇溶液上加入3毫升40%碘克沙醇溶液。然后将三毫升20%碘克沙醇溶液层在40%碘克沙醇溶液上。使用1,000微升移液管，在制备的20%碘克沙醇溶液上加入3毫升10%碘克沙醇溶液。接下来，在 10% 碘克沙醇溶液上加入 3.5 毫升 PBS/BEV 溶液，然后轻轻地将试管放回直立位置。现在，将一个 31 毫升的超速离心管放在带孔的泡沫板上，并用向上的箭头标记它。使用1,000微升移液管，将2.5毫升60%碘克沙醇储备溶液垂直加入管底部，并将其与500微升PBS/BEV溶液混合，得到3毫升50%碘克沙醇BEV溶液。通过移液彻底混合溶液。然后依次分层40%、20%和10%碘克沙醇溶液后，使用1000微升移液管在10%碘克沙醇溶液上加入3.5毫升PBS。用PBS将两根管子的重量调整为正负0.005克以内的总重量。接下来，将试管放在桶中，并在 4 摄氏度下以 160， 000 G 离心 7 小时。使用移液器，将从梯度超速离心模式获得的馏分依次从上到下沿侧壁收集到 10 个单独的 38.5 毫升超速离心管中。然后将馏分在 160， 000 G 下在 4 摄氏度下超速离心 70 分钟。除去上清液，将超速离心管倒置五分钟。接下来，使用200微升移液管将沉淀重悬于200微升预冷的PBS中。将 PBS/BEV 溶液转移到干净的 1.5 毫升微管中。标记从一种梯度超速离心模式获得的从F1到F10的馏分，然后进行分析。