Isolation and Characterization of USC-Derived Exosomes

USC-Exos were isolated from the culture supernatants according to published studies (13 (link), 14 (link)). After the cells were cultured with fresh complete proliferation medium containing 10% exosome-free FBS (Shanghai VivaCell Biosciences Ltd., China) for ~48 h, the medium was collected and centrifuged using an Amicon Ultra15 Centrifugal Filter Tube (10 kDa; Millipore). Finally, ~1 mL of ultrafiltration solution was mixed uniformly with ExoQuick-TC Solution (System Biosciences, USA) at a volume ratio of 5:1 by inversion. After centrifugation at 1,500 × g for 30 min, the exosome pellets in the bottom of the tube were resuspended in aseptic PBS based on the volume of the exosome pellets. The protein content of exosomes was measured using the BCA Assay Kit (Multi Sciences LTD., Hangzhou, China). The exosome solution was stored at −80°C until use in subsequent experiments.
The expression of TSG101 (ab125011; Abcam), CD63 (sc-5275; Santa), and calnexin (ab22595; Abcam) in isolated exosomes was determined by western blotting as described previously (14 (link)). USC extract was used as a control. The size distribution of USC-Exos was measured by DLS. The morphology of exosomes was observed by TEM (13 (link), 14 (link)).