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

Enhanced Bacterial Extracellular Vesicles Isolation for Microbiota Multi-Omics

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.

Author Spotlight: Accelerating Research on Bacterial Extracellular Vesicles Separation and Heterogeneity 

Isolation and Purification of Bacterial Extracellular Vesicles from Human Feces Using Density Gradient Centrifugation

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  

To begin, take 500 microliters of the prepared PBS/BEV solution. Combine it with three milliliters of PBS and mix gently using a 1, 000 microliter pipette. Place a 31 milliliter ultracentrifuge tube on a foam plate with holes, and label the tube with a downward arrow.Then hold a 1, 000 microliter pipette vertically and add three milliliters of the 50%iodixanol solution to the bottom of the tube. Place a tube holder slightly above the level of the foam plate below the tube opening, and tilt the tube to an angle of 70 degrees. Using a 1, 000 microliter pipette, add three milliliters of 40%iodixanol solution on top of the 50%iodixanol solution.Then layer three milliliters of 20%iodixanol solution on the 40%iodixanol solution. Using a 1, 000 microliter pipette, add three milliliters of a 10%iodixanol solution on top of the prepared 20%iodixanol solution. Next, add 3.5 milliliters of the PBS/BEV solution on top of the 10%iodixanol solution, and gently return the tubes to an upright position.Now, position a 31 milliliter ultracentrifuge tube on a foam plate with holes, and label it with an upward arrow. Using a 1, 000 microliter pipette, vertically add 2.5 milliliters of the 60%iodixanol stock solution into the bottom of the tube, and combine it with 500 microliters of PBS/BEV solution to get three milliliters of 50%iodixanol BEV solution. Mix the solution thoroughly by pipetting.Then after layering 40, 20 and 10%iodixanol solution in sequence, use a 1, 000 microliter pipette to add 3.5 milliliters of PBS on top of the 10%iodixanol solution. Adjust the weight of the two tubes with PBS to a total weight within plus minus 0.005 grams. Next, position the tubes in the buckets and centrifuge them at 160, 000 G for seven hours at four degrees Celsius.Using a pipetter, collect the fractions obtained from a gradient ultracentrifugation mode sequentially from top to bottom against the sidewall into 10 separate 38.5 milliliter ultracentrifuge tubes. Then ultracentrifuge the fractions at 160, 000 G for 70 minutes at four degrees Celsius. Remove the supernatant, and invert the ultracentrifuge tubes for five minutes.Next, resuspend the pellets in 200 microliters of pre-chilled PBS using a 200 microliter pipette. Transfer the PBS/BEV solution into a clean 1.5 milliliter microtube. Label the fractions from F1 to F10 as obtained from one gradient ultracentrifugation mode, and proceed for analysis.

establishment-density-gradient-centrifugation-system-to-isolate

Research

JoVE Journal

Biology

Bacterial extracellular vesicles (BEVs) are nanovesicles derived from bacteria that play an active role in bacteria-bacteria and bacteria-host communication, transferring bioactive molecules such as proteins, lipids, and nucleic acids inherited from the parent bacteria. BEVs derived from the gut microbiota have effects within the gastrointestinal tract and can reach distant organs, resulting in significant implications for physiology and pathology. Theoretical investigations that explore the types, quantities, and roles of BEVs derived from human feces are crucial for understanding the secretion and function of BEVs from the gut microbiota. These investigations also necessitate an improvement in the current strategy for isolating and purifying BEVs.
This study optimized the isolation and purification process of BEVs by establishing two density gradient centrifugation (DGC) modes: Top-down and Bottom-up. The enriched distribution of BEVs was determined in fractions 6 to 8 (F6-F8). The effectiveness of the approach was evaluated based on particle morphology, size, concentration, and protein content. The particle and protein recovery rates were calculated, and the presence of specific markers was analyzed to compare the recovery and purity of the two DGC modes. The results indicated that the Top-down centrifugation mode had lower contamination levels and achieved a recovery rate and purity similar to that of the Bottom-up mode. A centrifugation time of 7 h was sufficient to achieve a fecal BEV concentration of 108/mg.
Apart from feces, this method could be applied to other body fluid types with proper modification according to the differences in components and viscosity. In conclusion, this detailed and reliable protocol would facilitate the standardized isolation and purification of BEVs and thus, lay a foundation for subsequent multi-omics analysis and functional experiments.

This study describes a method to isolate and purify bacterial extracellular vesicles (BEVs) enriched from human feces via density gradient centrifugation (DGC), identifies the physical characteristics of BEVs from morphology, particle size, and concentration, and discusses the potential applications of the DGC approach in clinical and scientific research.

Bacterial extracellular vesicles (BEVs) are nanovesicles derived from bacteria that play an active role in bacteria-bacteria and bacteria-host ...

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

To begin, take the prepared human fecal samples. Cool the high speed refrigerated centrifuge to 4 degrees Celsius. Use PBS to adjust the weight of the sample tubes to 0.1 grams, and centrifuge them at 3, 000 G for 20 minutes at 4 degrees Celsius.Using a plastic pipette, transfer the supernatant into two 50 milliliter tubes, leaving 1 milliliter behind. Then, centrifuge the supernatant at 12, 000 G for 30 minutes at 4 degrees Celsius. Using a 20 milliliter syringe, aspirate the supernatant, and remove the needle from the syringe.Filter the liquid through 0.22 micrometer filters into 50 milliliter tubes. Next, wipe the bucket with 75%alcohol to eliminate any remaining contamination. Transfer approximately 30 milliliters of the filtered fecal supernatant into the ultracentrifuge tube.Fill the tube with approximately 8 milliliters of PBS, leaving a 3 millimeter gap from the tube's opening. Place the two ultracentrifuge tubes in opposing ultracentrifugation buckets, and equalize the weight of the two buckets using PBS. Load all buckets onto the rotor, irrespective of whether they are loaded with tubes.Place the samples in the centrifuge, initiate the vacuum and spin them at 160, 000 G for 70 minutes at 4 degrees Celsius. As soon as the centrifugation completes, release the chamber vacuum and open the door of the ultra centrifuge. Then, promptly remove the rotor.Transfer the buckets from the rotor to the rack, and pick up the tubes using a nipper. Discard the supernatant, and save the visible brown pellets. Add 1 milliliter of pre-chilled PBS to resuspend the pellets completely.Fill the tube with approximately 37 milliliters of PBS, leaving a 3 millimeter gap from the opening. After performing another round of ultracentrifugation, discard the supernatant and leave the tube inverted for 5 minutes. Remove any remaining solution on the inner wall with wipers.Using a 1, 000 microliter pipette, add 1.2 milliliters of pre-chilled PBS to resuspend pellets, and mix them by pipetting. Transfer 1.2 milliliters of the PBS BEV solution from one ultracentrifuge tube to a clean 1.5 milliliter micro tube.