Exosome precipitation kit

sEVs were isolated from 5 times concentrated MSCs-CM after the preliminary removal of cellular debris and large vesicles by centrifugation (3000× g for 15 min) with the Exosome Precipitation Kit (System Biosciences, Palo Alto, Canada), in accordance with the manufacturer’s recommendations. Briefly, prepared MSCs-CM was mixed with precipitation solution ExoQuick-TC (10 mL of MSCs-CM/2 mL of ExoQuickTC) and incubated overnight at 4 °C. After incubation, ExoQuick-TC/MSCs-CM mixtures were centrifuged at 1500× g for 30 min. Precipitated sEVs were diluted into sterile PBS and stored at −20 °C before the following experiments.

Exosomes were isolated as described previously39 (link). Briefly, conditioned medium was harvested 48 hours after cell seeding. After centrifugation at 3000 g to remove cellular debris, the supernatant was filtered through 0.22-μm PVDF filter (Millipore) to remove large vesicles. Exoquick Exosome Precipitation Solution (System Biosciences) was added to the filtered culture medium and mixed well. After refrigeration for 12 h, the mixture was centrifuged at 1500 g for 30 min. Exosome pellets were resuspended with PBS. The protein concentrations were measured by the Bradford method using a protein assay kit (Bio-Rad) as previously reported40 (link). To confirm the contents of the purified exosome samples, we performed immunoblotting to detect tetraspanin CD63 (Supplementary Fig. 6A). The size distribution of the exosomes in the culture supernatants was evaluated with the NanoSight LM10 system using Nanoparticle Tracking Analysis (NTA) software v2.3 (NanoSight Ltd, Amesbury, UK). The mean vesicle size in the samples was 89 nm (Supplementary Fig. 6B), in accordance with previous reports35 (link)41 (link). Purified exosomes were added to the culture medium at a concentration of 50 μg/ml. For plasma samples, exosome isolation was performed using ExoQuick Plasma prep and the Exosome precipitation kit (System Biosciences).

PRP-Exos were isolated from PRP through the combination of polymerization precipitation and ultracentrifugation. Crude PRP-Exos were initially precipitated via polymerization precipitation using ExoQuick (EXOQ5TM-1, ExoQuick Plasma prep and Exosome precipitation kit, System Biosciences, CA, USA). To enhance purity by removing impurities such as lipoproteins, we resuspended the PRP-Exos pellet in PBS and subjected it to ultracentrifugation (4 °C, 100,000×g, 70 min). The purified PRP-Exos pellet was subsequently stored at −80 °C.
The final size of exosome particles was determined through NTA using ZetaView (Particle Metrix, Meerbusch, Germany). Exosome morphology was characterized using transmission electron microscopy (Hitachi, H7500, Tokyo, Japan). The concentration of exosomes was quantified using a Pierce^TM BCA Protein Assay Kit (#23227, Thermo Scientific, Rockford, USA). The surface marker molecules of PRP-Exos were identified via western blot analysis, with primary antibodies including anti-CD9 (1:1000, 60232-1-Ig, Proteintech, USA), anti-CD63 (1:5000, 67605-1-Ig, Proteintech, USA), anti-CD81 (1:5000, 66866-1-Ig, Proteintech, USA), and anti-CD41 antibodies (1:4000, 60350-1-Ig, Proteintech, USA). All of the original western blot bands were provided in Supplementary Information (Supplementary Fig. 1).