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

Our study isolates, purifies, and characterizes bacterial extracellular vesicles from feces using density gradient centrifugation. We aim to adjust the separation of BEVs from contaminants and provide a reliable method for researchers. Our protocol reduces centrifugation time from 18 to seven hours.

Top-down mode improves BEV separation and reduces contamination from lipoproteins. 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.

To begin, take the prepared human fecal samples. Cool the high-speed refrigerated centrifuge to four degrees Celsius. Use PBS to adjust the weight of the sample tubes to 0.1 grams and centrifuge them at 3, 000G for 20 minutes at four degrees Celsius.

Using a plastic pipette, transfer the supernatant into two 50-milliliter tubes, leaving one milliliter behind. Then centrifuge the supernatant at 12, 000G for 30 minutes at four 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 eight milliliters of PBS, leaving a three 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, 000G for 70 minutes at four degrees Celsius. As soon as the centrifugation completes, release the chamber vacuum and open the door of the ultracentrifuge. 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 one milliliter of pre-chilled PBS to resuspend the pellets completely.

Fill the tube with approximately 37 milliliters of PBS, leaving a three-millimeter gap from the opening. After performing another round of ultracentrifugation, discard the supernatant and leave the tube inverted for five 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 microtube. 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, 000G for seven hours at four degrees Celsius. Using a pipettor, 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, 000G 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.