220 twin

The hucMSC-sEVs were isolated and purified as previously described [24 (link), 25 (link)]. The conditioned medium of P3-6 hucMSCs with good growth condition was collected. Cell supernatants were centrifuged to remove cell debris and organelles. Finally, the exosome pellets were resuspended in PBS and then passed through a 0.22-μm filter (Millipore, USA) and stored at − 80 °C. The protein content of the hucMSC-sEVs was determined by using a BCA protein assay kit (Vazyme, Nanjing, China). The morphology of the hucMSC-sEVs was observed using transmission electron microscopy (TEM; H-7800, Hitachi, Japan). The particle size, concentration, and zeta potential of the hucMSC-sEVs were analyzed by Nanoparticle tracking analyzer (NTA) (Germany, Particle Metrix, 220-Twin). The positive markers of hucMSC-sEVs, such as CD9, CD63, CD81, TSG101, Alix, and HSP70, as well as the negative control Calnexin, were determined by western blotting.

SEVs isolation was performed using an exosome isolation kit (UR52121, Umibio, China). After removing cell fragments, the supernatant was added exosome concentration solution (ESC) in the proportion of 4:1. After blending, the mixed liquid was placed at 4 °C for 2 h. Then, sEVs precipitation is separated out by centrifugation at 10000 × g for 1 h. Then, the PBS-washed sEVs were purified with an exosome purification filter at 3000 × g for 10 min at 4 °C. Lastly, the sEVs were stored at − 80 °C for further investigation. BCA protein assay kit (CW0014, CWBIO, China) was used to determine the protein concentration of sEVs. The marker protein CD9 (ab236630, Abcam, UK) was detected by western blot analysis. Transmission electron microscopy (TEM; H-7800, Hitachi, Japan) and nanosight tracking analysis (220-Twin, Particle Metrix, GER) were used to measure the appearance and size of sEVs.