This article discusses the extraction of extracellular vesicles (EVs) from whole tissue samples, highlighting their role in cell-to-cell communication. The methodology involves using a dissociation buffer and a series of centrifugation steps to isolate EVs effectively.
In whole tissues, extracellular vesicles or EVs are released by the cells into the interstitial fluid. These include apoptotic bodies, microvesicles, and exosomes. The EVs play an important role in maintaining cell-to-cell communication.
To extract EVs, begin by taking a whole tissue sample.
Treat the tissue with an appropriate dissociation buffer. The components of dissociation buffer disintegrate the extracellular matrix present in the tissue, eventually loosening the cells.
Transfer the suspension to a homogenizer and mechanically disrupt the tissue further to dissociate it and release the cells.
Next, centrifuge the homogenate at low-speed to pelletize the cells and tissue fragments while the EVs remain in the supernatant.
Take the supernatant into a fresh tube. Centrifuge at high speed to pelletize most of the larger-sized apoptotic bodies, leaving the smaller-sized EVs in the supernatant.
Collect the EV-containing supernatant into a syringe. Filter the components to remove any remaining apoptotic bodies and collect the small-sized EV population.
Centrifuge the filtrate to obtain the EVs in the pellet. Discard the remaining buffer and resuspend the EV pellet in a sucrose solution. This solution creates an isotonic environment for the EVs to retain their morphology.
Now, store the EV suspension at four degrees Celsius until further use.
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.
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