Isolation and Culture of Vascular Cells

Pooled HUVECs (C-12208, lot 447Z015; Promocell, Heidelberg, Germany) were cultivated in endothelial cell growth medium MV2 supplemented with 5% (v/v) fetal bovine serum, 5 ng/mL epidermal growth factor, 10 ng/mL basic fibroblast growth factor, 20 ng/mL insulin-like growth factor-1, 0.5 ng/mL vascular endothelial growth factor 165, 1 µg/mL ascorbic acid, and 0.2 µg/mL hydrocortisone (Promocell, Heidelberg, Germany). HSVSMCs were isolated from surplus vein tissue from consenting subjects undergoing coronary artery bypass graft surgery as previously described [32 (link)] and cultivated in smooth muscle cell growth medium 2 supplemented with 5% (v/v) fetal bovine serum, 0.5 ng/mL epidermal growth factor, 2 ng/mL basic fibroblast growth factor, and 5 µg/mL insulin (Promocell, Heidelberg, Germany). SMC integrity was verified by the presence of SMC markers myosin heavy chain and α-actin and the absence of the endothelial marker PECAM1 (CD31). Cultures were maintained at 37 °C in a humidified atmosphere containing 5% (v/v) CO₂.