Adipose-derived MSCs Glucose Adaptation

Human adipose-tissue derived MSCs (hAD-MSCs) were commercially purchased from Lonza (Cat# PT5006, Lot# 21TL138912) and expanded using Dulbecco’s Modified Eagle’s Medium low glucose (DMEM-low glucose, Euroclone), which contained 5.6 mmol/L glucose and were supplemented with 10% fetal bovine serum (FBS, Gibco)[19 (link)], 0.1 mg/mL streptomycin and 100 units/mL penicillin G in standard cell culture incubators (5% CO₂/95% air; 37 °C). Medium was changed every 72 h and cells were sub-cultured when confluence exceeded 60%. For high glucose conditions, cells were cultured in DMEM high glucose (DMEM-High Glucose, Euroclone) which contained 25 mmol/L glucose and were supplemented with 10% FBS, 0.1 mg/mL streptomycin and 100 units/mL penicillin G for 5 d in standard cell culture incubators (5% CO₂/95% air; 37 °C). High glucose complete medium was changed every 72 h. For the preparation of the high glucose media, we followed a previously reported protocol[20 (link),21 ]. MSCs that were cultured in low glucose were considered as our study control. MSCs of passage 6 (P6), P8, and P10 were used to perform the experiments.