Name: Bacterial Extracellular Vesicles (BEVs) Enrichment from Human Faeces Using Differential Centrifugation Speeds
Uploaded: 2023-09-01T14:30:00
Description: Watch this Scientific Journal Video about Bacterial Extracellular Vesicles BEVs Enrichment from Human Faeces Using Differential Centrifugation Speeds at JoVE.com

bacterial extracellular vesicles (bevs) enrichment from human faeces using differential centrifugation speeds

Mikrobiyota Multi-Omikler için Gelişmiş Bakteriyel Hücre Dışı Vezikül İzolasyonu

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.

Yazar Spotlight: Bakteriyel Hücre Dışı Veziküllerin Ayrılması ve Heterojenliği Üzerine Araştırmaların Hızlandırılması 

Yoğunluk Gradyanlı Santrifüj Kullanılarak İnsan Dışkısından Bakteriyel Hücre Dışı Veziküllerin İzolasyonu ve Saflaştırılması

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 Bacterial Extracellular Vesicles BEVs Enrichment from Human Faeces Using Differential Centrifugation Speeds at JoVE.com

Bacterial Extracellular Vesicles (BEVs) Enrichment from Human Faeces Using Differential Centrifugation Speeds  

Diferansiyel Santrifüj Hızları Kullanılarak İnsan Dışkısından Bakteriyel Hücre Dışı Veziküllerin (BEV'ler) Zenginleştirilmesi

Başlamak için hazırlanan insan dışkı örneklerini alın. Yüksek hızlı soğutmalı santrifüjü 4 santigrat dereceye soğutun. Numune tüplerinin ağırlığını 0.1 grama ayarlamak için PBS kullanın ve bunları 4 santigrat derecede 20 dakika boyunca 3.000 G'de santrifüjleyin.Plastik bir pipet kullanarak, süpernatanı 50 mililitrelik iki tüpe aktarın ve geride 1 mililitre bırakın. Daha sonra, süpernatanı 12.000 G'de 4 santigrat derecede 30 dakika santrifüjleyin. 20 mililitrelik bir şırınga kullanarak, süpernatanı aspire edin ve iğneyi şırıngadan çıkarın.Sıvıyı 0.22 mikrometre filtrelerden 50 mililitrelik tüplere süzün. Ardından, kalan kontaminasyonu ortadan kaldırmak için kovayı %75 alkolle silin. Filtrelenmiş fekal süpernatanın yaklaşık 30 mililitresini ultrasantrifüj tüpüne aktarın.Tüpü yaklaşık 8 mililitre PBS ile doldurun ve tüpün açıklığından 3 milimetre boşluk bırakın. İki ultrasantrifüj tüpünü karşılıklı ultrasantrifüj kovalarına yerleştirin ve PBS kullanarak iki kovanın ağırlığını eşitleyin. Borularla yüklü olup olmadıklarına bakılmaksızın tüm kovaları rotora yükleyin.Numuneleri santrifüje yerleştirin, vakumu başlatın ve 4 santigrat derecede 70 dakika boyunca 160.000 G'de döndürün. Santrifüjleme tamamlanır tamamlanmaz, hazne vakumunu serbest bırakın ve ultra santrifüjün kapısını açın. Ardından, rotoru hemen çıkarın.Kovaları rotordan rafa aktarın ve boruları bir pense kullanarak alın. Süpernatanı atın ve görünür kahverengi topakları saklayın. Peletleri tamamen yeniden süspanse etmek için 1 mililitre önceden soğutulmuş PBS ekleyin.Tüpü yaklaşık 37 mililitre PBS ile doldurun ve açıklıktan 3 milimetre boşluk bırakın. Başka bir ultrasantrifüj turu gerçekleştirdikten sonra, süpernatanı atın ve tüpü 5 dakika ters çevirin. İç duvarda kalan çözeltiyi silecekler ile çıkarın.1.000 mikrolitrelik bir pipet kullanarak, peletleri yeniden süspanse etmek için 1,2 mililitre önceden soğutulmuş PBS ekleyin ve pipetleyerek karıştırın. 1.2 mililitre PBS BEV çözeltisini bir ultrasantrifüj tüpünden temiz bir 1.5 mililitre mikro tüpe aktarın.