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.