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

Aislamiento mejorado de vesículas extracelulares bacterianas para microbiota multiómica

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.

Autor destacado: Aceleración de la investigación sobre la separación y heterogeneidad de vesículas extracelulares bacterianas 

Aislamiento y purificación de vesículas extracelulares bacterianas a partir de heces humanas mediante centrifugación en gradiente de densidad

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  

Establecimiento de un sistema de centrifugación en gradiente de densidad para aislar y purificar vesículas extracelulares bacterianas (BEV) de heces humanas

Para comenzar, tome 500 microlitros de la solución preparada de PBS/BEV. Combínalo con tres mililitros de PBS y mezcla suavemente con una pipeta de 1.000 microlitros. Coloque un tubo de ultracentrífuga de 31 mililitros en una placa de espuma con agujeros y etiquete el tubo con una flecha hacia abajo.A continuación, sostenga una pipeta de 1.000 microlitros verticalmente y añada tres mililitros de la solución de yodixanol al 50% en el fondo del tubo. Coloque un soporte para tubos ligeramente por encima del nivel de la placa de espuma debajo de la abertura del tubo e incline el tubo en un ángulo de 70 grados. Con una pipeta de 1.000 microlitros, agregue tres mililitros de solución de yodixanol al 40 % sobre la solución de iodixanol al 50 %.A continuación, coloque tres mililitros de solución de yodixanol al 20% sobre la solución de yodixanol al 40%. Con una pipeta de 1.000 microlitros, añadir tres mililitros de una solución de yodixanol al 10 % encima de la solución preparada de yodixanol al 20 %. A continuación, agregue 3,5 mililitros de la solución de PBS/BEV encima de la solución de yodixanol al 10% y vuelva a colocar suavemente los tubos en posición vertical.Ahora, coloque un tubo de ultracentrífuga de 31 mililitros en una placa de espuma con agujeros y etiquételo con una flecha hacia arriba. Con una pipeta de 1.000 microlitros, agregue verticalmente 2,5 mililitros de la solución madre de yodixanol al 60 % en el fondo del tubo y combínelo con 500 microlitros de solución de PBS/BEV para obtener tres mililitros de solución de BEV al 50 % de iodixanol. Mezcle bien la solución pipeteando.A continuación, después de aplicar una solución de 40, 20 y 10 % de yodixanol en secuencia, utilice una pipeta de 1.000 microlitros para añadir 3,5 mililitros de PBS encima de la solución de 10 % de iodixanol. Ajuste el peso de los dos tubos con PBS a un peso total dentro de más menos 0,005 gramos. A continuación, coloque los tubos en los cubos y centrifugérelos a 160.000 G durante siete horas a cuatro grados centígrados.Con una pipeta, recoja las fracciones obtenidas de un modo de ultracentrifugación de gradiente secuencialmente de arriba a abajo contra la pared lateral en 10 tubos de ultracentrífuga separados de 38,5 mililitros. A continuación, ultracentrifugar las fracciones a 160.000 G durante 70 minutos a cuatro grados centígrados. Retire el sobrenadante e invierta los tubos de ultracentrífuga durante cinco minutos.A continuación, vuelva a suspender los gránulos en 200 microlitros de PBS preenfriado con una pipeta de 200 microlitros. Transfiera la solución de PBS/BEV a un microtubo limpio de 1,5 mililitros. Etiquete las fracciones de F1 a F10 como obtenidas de un modo de ultracentrifugación de gradiente y proceda al análisis.