Exosome isolation and purification

MSCs were cultured in media supplemented with 10% exosome-depleted FBS (FBS, Gibco). The depletion of bovine exosomes from FBS was achieved by ultracentrifugation at 100,000g for 70 min. Rat exosomes were collected from 24-hour culture in conditioned media through standard differential centrifugation steps. The cell culture supernatant was collected and the exosomes isolated by centrifugation at 2000 x g for 20 min then by pelleting using ultracentrifugation at 100,000 x g for 1 h at 4℃. Finally, the exosome pellet was washed in a large volume of PBS then resuspended in PBS. The exosomes were further purified by resuspending in 2.5 M sucrose in 25 mM HEPES buffer (pH 7.4). They were then subsequently loaded into the bottom of a SW41 tube. HEPES buffer (25mM) containing 2 M sucrose was carefully loaded on top of the exosomes followed by HEPES buffer (25 mM) containing 0.25 M sucrose to produce a discontinuous 2-0.25 M sucrose gradient. After spinning overnight at 100,000 x g in an SW41 swing rotor, 1 mL of each fraction was collected then centrifuged at 100,000 x g for 1 h. After aspirating the supernatant, the pellet was resuspended in PBS, the protein content quantified using a bicinchoninic acid (BCA) assay (Thermo Fisher Scientific Inc.) and then stored at -80℃ until required for use.