Isolation and Purification of Exosomes
I. Purifying exosomes from cell cultures
The general idea of exosome purification by ultracentrifugation is depicted in the following flow chart. The first steps are designed to eliminate large dead cells and large cell debris by successive centrifugations at increasing speeds (steps 1 to 5 below). At each of these steps, the pellet is thrown away, and the supernatant is used for the following step (see the following flow chart). The final supernatant is then ultracentrifuged at 100,000×g to pellet the small vesicles that correspond to exosomes. The pellet is washed in a large volume of PBS, to eliminate contaminating proteins, and centrifuged one last time at the same high speed.
Materials
*Conditioned medium (Support Protocol 1, step 5), cleared
*Phosphate-buffered saline (PBS);
*Tris-buffered saline (TBS);
*Refrigerated centrifuge;
*50-ml polypropylene centrifuge tubes;
*Ultracentrifuge and fixed-angle or swinging-bucket rotor;
*Polyallomer tubes or polycarbonate bottles, appropriate for the ultracentrifuge
Rotor;
*Micropipettor (e.g., Pipetman)
*Tabletop ultracentrifuge (e.g., Beckman TL-100) –80°C freezer.
NOTE: All centrifugations should be performed at 4°C.
Procedures:
Remove cells, dead cells, and cellular debris
1. Transfer the cleared, conditioned medium to 50-ml centrifuge tubes.
2. Centrifuge 20 min at 2,000×g, 4°C.
3. Pipet off the supernatant, and transfer it to polyallomer tubes or polycarbonate bottles appropriate for the ultracentrifugation rotor to be used.
4. Mark one side of each ultracentrifuge tube with a waterproof marker, orient the tube in the rotor with the mark facing up, and centrifuge 30 min at 10,000×g, 4°C.
Collect exosome fraction:
5. Transfer the supernatant to a fresh tube or bottle the same size as in step 3.
6. Centrifuge at least 70 min at 100,000×g, 4°C.
7. Remove the supernatant completely.
Wash exosomes:
8. Resuspend the pellet in each tube in 1 ml PBS, using a micropipettor. Pool the resuspended pellets from all the tubes containing materials from the same cells in a single centrifuge tube. Then add PBS to fill the tube completely.
9. Centrifuge 1 hr at 100,000×g, 4°C.
10. Remove the supernatant as completely as possible. For fixed-angle rotors, pour off the supernatant, keep the tube upside down, and aspirate the remaining liquid on the sides and the mouth of the tube with a micropipettor. Proceed to step 11a or step 11b.
11a:
To resuspend the pellet (i.e., exosomes): Add a small volume (50 to 100μl) of PBS or TBS and resuspend. If the final volume of exosomes is too large (>1/2000 of the initial volume of conditioned medium) or if there was no visible pellet at step 10, use step 11b instead.
11b:
To concentrate the exosome preparation: Centrifuge the supernatant (step 10) 1 hr at 100,000×g, 4°C in a tabletop ultracentrifuge, using a TLA-100.3 rotor and the corresponding thick-walled polycarbonate tubes. Remove most of the PBS above the visible pellet and resuspend exosomes in 20 to 50μl of fresh PBS.
12. Store exosomes up to 1 year at –80°C in 100-μl aliquots. Avoid repeated freezing and thawing.
II. Purifying exosomes from viscous fluids
Materials
*Fluid (e.g., plasma: separate from blood cells by Ficoll centrifugation; lymph, serum, urine, bronchiolar lavage, or tumor ascites);
*Phosphate-buffered saline (PBS);
*Refrigerated centrifuge;
*50-ml polypropylene centrifuge tubes;
*0.22-μm filter device (e.g., Steritop, Millipore);
*Ultracentrifuge and fixed-angle or swinging-bucket rotor;
*Polyallomer tubes or polycarbonate bottles, appropriate for the ultracentrifuge
Rotor.
NOTE: All centrifugations should be performed at 4°C.
1. Dilute fluid with an equal volume of PBS. Transfer diluted fluid in 50-ml tubes. Centrifuge 30 min at 2,000×g, 4°C.
2. Carefully transfer supernatant to ultracentrifuge tubes or bottles without pellet contamination. Centrifuge 45 min at 12,000×g, 4°C.
3. Carefully transfer supernatant to ultracentrifuge tubes or bottles for tubes to choose according to the volume of fluid handled). Centrifuge 2 hours at 110,000×g, 4°C.
4. Resuspend pellets in 1 ml PBS and pool them in one of the tubes. Fill the tube with PBS to dilute the resuspension in a large volume.
5. Filter the suspension through a 0.22-μm filter, and collect in a fresh ultracentrifuge tube or bottle.
6. Centrifuge 70 min at 110,000×g, 4°C. Pour off the supernatant.
7. Resuspend the pellet in 1 ml PBS, and then fill the tube with PBS. Centrifuge 70 min at 110,000×g, 4°C. Pour off the supernatant.
8. Resuspend pellet in 50 to 200μl PBS. Store up to1year at−80°C.
