Extracellular Vesicle Extraction: A Technique to Isolate Extracellular Vesicles from Whole Tissue Specimens Using Differential Centrifugation

To begin, prepare 10 milliliters of dissociation buffer in Hibernate-E Medium for every 0.4 to 1.0 gram of tissue. Add whole fresh or frozen tissue to the buffer in a 50-milliliter tube and incubate in a warm water bath at 37 degrees Celsius for 20 minutes.

After this, add protease and phosphatase inhibitors for a final 1x concentration in the dissociation buffer. Pour the solution with the tissue into a loose-fit Dounce homogenizer. Use approximately 30 slow strokes per sample to gently dissociate the tissue.

Then, transfer the dissociated tissue and buffer solution to a 50-milliliter conical tube. Centrifuge at 500 x g and at 4 degrees Celsius for 5 minutes to pellet the cells and the remaining fibrous or cohesive tissue fragments.

Transfer the supernatants to a clean 50-milliliter conical tube and centrifuge at 2,000 x g and at 4 degrees Celsius for 10 minutes to pellet and discard the large cellular debris.

Transfer this supernatant to a clean 50-milliliter conical tube and centrifuge at 10,000 x g and at 4 degrees Celsius for 40 minutes to pellet any undesired larger vesicles or small apoptotic bodies. Decant the supernatant through a 0.45-micrometer filter into a clean 12-milliliter ultracentrifugation tube.

Next, ultracentrifuge the sample at 100,000 x g and at 4 degrees Celsius for 2 hours to pellet small EVs. Decant the supernatant and leave the ultracentrifugation tubes inverted for 5 to 10 minutes, tapping frequently to remove any residual liquid on the sides of the tubes.

Then, resuspend the EV pellet in 1.5 milliliters of 0.25-molar sucrose buffer. Cover the tubes with parafilm and then vortex the EVs into solution. Rock the ultracentrifuge tubes for 15 to 20 minutes at room temperature and then vortex once more.

Briefly centrifuge the tubes at a speed less than 1,000 x g to recover the liquid suspension at the bottom of the tube. If needed, store the suspension at 4 degrees Celsius overnight.