32 ti rotor

The exosomes were purified by sequential centrifugation as previously described [44 (link),62 (link),63 (link)]. In brief, cells were cultured in media supplemented with 10% exosome-free FBS. After 72 h, cells were digested and counted. The supernatants were centrifuged at 500× g at 4 °C for 10 min to remove living cells. To remove dead cells and large cell debris, the supernatants were then centrifuged at 2000× g at 4 °C for 10 min. To remove large vesicles, the supernatants were centrifuged at 10,000× g at 4 °C for 30 min. Finally, the exosomes were harvested by centrifugation at 100,000× g at 4 °C for 70 min (Beckman 32 Ti rotor, Germany). The exosomes were washed with PBS (Thermo, USA) and pelleted again through ultracentrifugation at 100,000× g at 4 °C for 70 min. The exosome pellets were resuspended in the corresponding volume of PBS according to cell numbers to ensure that the same volume of PBS contained exosomes from the same cell number. Exosome size and particle concentration were analyzed using an NS300 nanoparticle characterization system (NanoSight, Malvern Instruments, Malvern, UK) equipped with a red laser (580 nm). The exosomes were lysed with denaturing RIPA buffer (plus the cocktail), and their markers were analyzed by WB. The exosomes were stored short-term at 4 °C and long-term at −80 °C.

After reaching 80–90% confluence, hADSCs were rinsed in dPBS (SH30028.02, HyClone, South Logan, UT, USA) and inoculated with a freshly prepared culture medium for two days. After that, isolation of exosomes from the hADSCs culture medium by ultracentrifugation. The culture medium of hADSCs was centrifuged at 2000 × g for 30 min at 4℃ to get rid of dead cells along with the debris, and then the supernate was transferred to a new 50 ml centrifuge tube, centrifuged at 10,000 × g for 30 min at 4℃ to get rid of other large vesicles. The supernatant would be collected again, put into a new 32ti rotor(Beckman Coulter, Miami, FL, USA), centrifuged at 100,000 × g for 2 h 15 min at 4℃. We removed the supernate, re-suspended the pellets in dPBS, and kept them at − 80 °C. The BCA protein assay kit (23,225, Thermo Fisher Scientific, Waltham, MA, USA) was employed to determine the protein quantitation of hADSCs exosomes. Conventional transmission electron microscopy coupled with NanoSight and Western blotting were adopted to explore hADSCs-Exos morphology, size, and marker expression (CD9, CD63, and CD81).

Exosomes were labeled using the red fluorescent dye PKH26 according to the manufacturer’s instructions (Sigma-Aldrich, USA). Excess dye from the labeled exosomes was removed by ultracentrifugation at 100,000g for 1 h at 4 °C using a 32 Ti rotor (Beckman Coulter, USA), and the exosome pellets were washed three times by PBS. The final pellets were resuspended in PBS. Exosomes were co-cultured with rat chondrocytes at a concentration of 10 μg/ml in serum-free medium at 37 °C for 12 h and then fixed with 4% paraformaldehyde. The nuclei were stained with Hoechst 33342 (10 μg/ml, Beyotime, China). The cytoskeleton was stained by Actin-Tracker Green (Beyotime, China). The uptake of exosome was observed using a confocal laser scanning microscope (Zeiss LSM710, Germany).
For the evaluation of exosome uptake in vivo, labeled exosomes (40 μg/100 μl) were injected into the joint cavity after the rat model of OA was established. Small animal fluorescence imager (eXplore Optix, Advanced Research Technology, USA) was used to monitor the signals in exosomes.