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

1. Tissue Dissociation and Differential Centrifugation

1. Prepare 10 mL of dissociation buffer (10 mg of papain; 5.5 mM L-cysteine; 67 µM 2-mercaptoethanol; 1.1 mM EDTA) in Hibernate-E medium for approximately 0.4–1.0 g of tissue.
   NOTE: All the solutions used for EV enrichment and purification should be diluted in ultrapure filtered water.
2. Add whole fresh or frozen tissue to dissociation buffer in a 50 mL conical tube and incubate in a warm water bath at 37 °C for 20 min. Tissue may be cut into smaller fragments before incubation if needed.
3. Following the incubation, add protease and phosphatase inhibitors for a final 1x concentration to the dissociation buffer containing the tissue.
4. Pour the solution containing the tissue into a loose-fit Dounce homogenizer, and gently dissociate the tissue using approximately 30 slow strokes per sample. The number of strokes may be adjusted based on tissue consistency.
5. Transfer dissociated tissue in buffer to a 50 mL conical tube and centrifuge at 500 x g for 5 min at 4 °C to pellet the cells and remaining fibrous or cohesive tissue fragments.
6. Transfer the supernatant to a clean 50 mL conical tube and centrifuge at 2,000 x g for 10 min at 4 °C to pellet and discard large cellular debris.
7. Transfer the supernatant again to a clean 50 mL conical tube and centrifuge at 10,000 x g for 40 min at 4 °C to pellet undesired larger vesicles or small apoptotic bodies.
   NOTE: This pellet can be saved for additional study of larger vesicles if desired.
8. Decant the supernatant through a 0.45 µm filter into a clean 12 mL ultracentrifugation tube.
   NOTE: Densely fibrotic tissue samples may not be easily filtered. These samples can be filtered through a 40 µm cell strainer, then passed through serially smaller needles (18 G, 20 G, 22 G) before filtering through the 0.45 µm filter.
9. Ultracentrifuge the sample at 100,000 x g for 2 h at 4 °C to pellet small EVs.
10. Decant the supernatant and leave the ultracentrifugation tubes inverted for 5–10 min, tapping frequently to remove residual liquid on the sides of tubes. Residual fluid can also be aspirated using an aspirating vacuum pipette.
11. Re-suspend EV pellet in 1.5 mL of 0.25 M sucrose buffer (10 mM Tris, pH 7.4). It is important to ensure full re-suspension of the pellet in this step. To do so, cover the tubes with parafilm before vortexing EVs into solution. Rock the ultracentrifuge tubes for 15-20 min at room temperature before a final vortex mix.
12. Briefly centrifuge the tubes at a speed no more than 1,000 x g to recover the liquid suspension at the bottom of the tube.
13. Proceed to the gradient purification steps below, or if needed, store EV suspension at 4 °C overnight.