Exosome Isolation: A Density-based Ultracentrifugation Technique Using Sucrose Cushion to Separate Exosomes from Conditioned Media of Cultured Cells

Exosomes - nano-sized, membrane-bound vesicles that carry functional biomolecules -  are actively secreted by most cell types and can be isolated from in vitro cell culture supernatants. To isolate exosomes, first, harvest the supernatant or conditioned media from a cell culture of interest. The conditioned media would contain exosomes, proteins, cellular debris, and vesicles.

Centrifuge the media to pellet the cellular debris. Collect the exosome-containing supernatant and filter it through cell strainers to remove the larger vesicles. Transfer a specific volume of the filtered media to a fresh tube. Next, mix appropriate concentrations of sucrose and deuterium oxide to prepare a sucrose cushion - a discontinuous gradient of sucrose. 

Add a specific volume of the sucrose cushion solution into the media-containing tube; the solution forms a separate layer at the bottom. Now, centrifuge the tube at high speed. The sucrose cushion facilitates the migration of exosomes from the media to the sucrose solution while maintaining exosome integrity. The high-density protein contaminants occupy the interface between the sucrose solution and the media.

Carefully transfer the major fraction of exosome-containing sucrose layer into a fresh tube with a suitable buffer. Centrifuge at high speed to obtain a pellet of purified exosomes. Discard the sucrose-containing supernatant along with any protein contaminants. Resuspend the exosome pellet in buffer and store at low temperatures until further use.

To pre-clear the collected conditioned medium, centrifuge it at 500 x g for 5 minutes at 4 degrees Celsius. Transfer the supernatant into a new tube and discard the pellet. After repeating this centrifugation step with the recovered supernatant, recover the supernatant again and discard the pellet.

Then, centrifuge the recovered supernatant at 2,000 x g for 15 minutes and 4 degrees Celsius. Keep the supernatant and discard the pellet. Filter the supernatant once through 0.22-micrometer filter attached to a 20-milliliter syringe into a fresh centrifuge tube.

Meanwhile, to prepare 25% sucrose solution in deuterium oxide, accurately weigh out 1.9 grams of sucrose in a universal tube. Then, add deuterium oxide until the weight reaches 7.6 grams. Then, fill up an ultracentrifuge tube with 22.5 milliliters of pre-cleared conditioned medium.

Place a glass pipette in the tube. Add 3 milliliters of sucrose solution through the pipette so that the solution forms a separate layer beneath the conditioned medium. Carefully place the tube containing layered conditioned medium sucrose solution into the bucket of a swing-out rotor.

Secure the bucket into the rotor. Place the rotor into the ultracentrifuge and spin it 100,000 x g at 4 degrees Celsius for 1.5 hours. Collect 2 milliliters of the sucrose layer from the tube, 1 milliliter at a time, using a P1000 pipette with its tip attached to a 10-microliter tip and add it to an ultracentrifuge bottle containing 20 milliliters of filtered PBS for washing.

Place the tube into a fixed-angle rotor and centrifuge it at 100,000 x g at 4 degrees Celsius for 1.5 hours. Use a 10-milliliter serological pipette to carefully remove the supernatant. Resuspend the pellet with 400 microliters of filtered PBS.