Cell culture

Mouse MSCs were purchased from Cyagen Biosciences (Guangzhou, China). The cells were maintained in a culture medium in a humidified incubator with 5% carbon dioxide (CO₂) at 37 °C, and routinely detected and verified to be free of mycoplasma contamination. Before collecting the cell culture supernatant for exosome isolation, fetal bovine serum (FBS, Biological Industries, Beit-Haemek, Israel) was first depleted of exosomes. The exo-depleted FBS was prepared by ultracentrifugation at 120,000 ×g for 18 h at 4 °C, and then, the supernatant was filtered through a 0.22-µm filter unit. Complete culture medium was prepared by supplementing 500 mL of Dulbecco’s modified eagle medium (DMEM) (Gibco, Grand Island, NY, USA) with 50 mL of exo-depleted FBS, 5 mL of 100× L-glutamine (Amresco, Dallas, TX, USA), and 2.5 mL of 100× Penicillin-Streptomycin Solution (Solarbio, Beijing, China). Approximately 3×10⁶ C57BL/6 MSCs in 30 mL of complete medium were seeded into each P150 tissue culture plate. The cells were cultured for 72 h before supernatant collection.