Co-Culture and sEV Transfer in OA-FLS and HDMECs

OA-FLS (4 × 10⁵ cells per ml) and HDMECs (3 × 10⁵ cells per ml) were co-cultured in the aforementioned transwell apparatus, followed by incubation in cell growth medium with depleted sEV (SBI). Subsequently, SMSCs-derived sEV (SMSCs-sEV, 100 µg) were put into the coculture. After incubation in fetal bovine serum (FBS), 1% Dulbecco modified Eagle medium for 48 h, the supernatants were obtained and centrifuged (120,000 × g, 120 min) to remove sEV. Subsequently, TFA together with ex vivo ARA of angiogenesis was performed under the management of culture supernatants. The remaining supernatants were further analyzed for VEGF using a commercial ELISA kit (R&D Systems). Furthermore, the expression of mRNA and protein in the OA-FLS and HDMECs coculture was measured by RT-qPCR as well as WB, respectively.
The PKH67 Green Fluorescent Cell Linker Kit (Sigma) was applied for the staining of the sEV as previously described (Zhang et al., 2021b (link)). Briefly, sEV were mixed and labeled with 100 μl PKH67 dye solution for 5 min, followed by incubation with recipient OA-FLS or HDMECs for 6 h using a laser confocal microscope (LEICA TCS SP8) before imaging.