Myocardial Ischemia/Reperfusion Injury Model

Myocardial cell H/R treatment was established in H9c2 myocardial cells (from the Cell Bank of the Chinese Academy of Sciences, Shanghai, China) to simulate I/R injury model. For hypoxic incubation, H9c2 cells were cultured in glucose-free and serum-free DMEM medium and exposed to a humidified incubation chamber flushed with a gas mixture of 95% N₂ and 5% CO₂ at 37°C. After 6 hours of hypoxic incubation, the cells were transferred to high glucose DMEM medium containing 10% FCS (reoxygenation medium), and reoxygenated in 95% air and 5% CO₂ at 37°C for 24 hours H9c2 cells were randomly divided into 5 groups^{1 (link)}: control group, cells cultured in 10% FBS-containing DMEM medium under normoxic conditions² ; H/R group, cells subjected to H/R injury only^{3 (link)}; H/R+PBS group, cells subjected to H/R injury and 200 μL exosome-free PBS was added to the reoxygenation medium^{4 (link)}; H/R+ADMSCs-ex group, cells subjected to H/R injury and 200 μL PBS containing 400 μg of ADMSCs-ex were added to the reoxygenation medium; and^{5 (link)} H/R+ADMSCs-ex+XAV939 group, cells subjected to H/R injury and 200 μL PBS containing 400 μg of ADMSCs-ex and Wnt/β-catenin inhibitors XAV939 (10 μM; Selleck, Houston, TX) were added to the reoxygenation medium.