Cushioned-Density Gradient Ultracentrifugation (C-DGUC) of Exosomes

Exosomes represent one class of extracellular vesicles that are thought to be shed by all cell types. Although the exact nature of exosome biogenesis and function remains incompletely understood, they are increasingly recognized as a source of intercellular communication in health and disease. Recent observations of RNA exchange via donor cell-derived exosomes that exert genetic regulation in recipient cells have led to a boon into exosome research. The excitement and promise of exosomes as a new therapeutic avenue for human pathologies remain limited by challenges associated with their isolation from culture media and biofluids. The introduction of new methodologies to facilitate the isolation of exosomes has simultaneously raised concerns related to the reproducibility of studies describing exosome effector functions. Even high-speed ultracentrifugation, the first and long considered gold standard approach for exosome isolation has recently been noted to be subject to uncontrolled variables that could impact functional readouts of exosome preparations. Researchers from UCSF describe principles and methods that attempt to overcome such limitations by first concentrating exosomes in a liquid cushion and subsequently resolving them using density gradient ultracentrifugation. This approach avoids possible complications associated with direct pelleting onto plastic tubes and allows for further purification of exosomes from dense protein aggregates.

Schematic illustration of C-DGUC for exosome isolation

(1). Place a 2 mL solution of 60% iodixanol below the pre-cleared conditioned medium that serves to cushion the nanoparticles during centrifugation; (2) sediment the nanoparticles at 100,000 × g for 3 h; (3) remove the 2 mL cushion along with 1 mL of overlaying medium and place it below a pre-formed step density gradient composed of three layers of iodixanol solution, 3 mL each; (4) perform density gradient ultracentrifugation at 100,000 × g for a period of 18 h; (5) collect the medium from the top of the tube in 1 mL increments. Exosomes derived from cultured primary BMDM typically are isolated in fractions 6 and 7