Isolation and Expansion of Human Adipose-Derived MSCs

Human MSCs were isolated from AT as previously described [9 (link), 10 (link)]. Briefly, subcutaneous AT samples were washed with phosphate buffered saline (PBS) (Wisent Inc., St. Bruno, QC) and minced with surgical scissors. The tissue was digested with 0.05% collagenase (Sigma‐Aldrich Corporation, MO, USA) dissolved in Hank’s balanced salt solution (Invitrogen, MA, USA). After 2 h at 37C, the collagenase was neutralized with the addition of fetal bovine serum (FBS), and samples were centrifuged (5 min at 2000 rpm). The pellet was resuspended in complete low glucose Dulbecco’s modified Eagle’s medium (DMEM) (Wisent Inc., St. Bruno, QC), supplemented with 10% MSC-qualified FBS (certified FBS-MSC, Gibco Invitrogen) and 1% penicillin/streptomycin (Wisent Inc., St. Bruno, QC). Digested tissue samples were cultured in T75 flasks at 37 °C in 5% CO₂ (1 g of tissue/flask), and non-adherent cells were washed off two days following isolation. Isolated MSC(AT) were seeded at a density of 4,000 cells/cm² in T75 flasks in complete medium and passaged at 80% confluency. Early and late passage MSC(AT) were defined as ≤ 5 and ≥ 15 passages respectively [11 (link), 12 (link)].