Human umbilical vein endothelial cells (abbreviated as HUVECs) were isolated, cultured, and characterized as previously described [44 (link),45 (link)]. Cells were cultured on gelatin-coated dishes in cM199 (M199 medium supplemented with 20% heat-inactivated new born calf serum (both from Cambrex, Verviers, Belgium), 150 μg/mL crude endothelial cell growth factor (ECGF), 5 U/mL heparin, 100 IU/mL penicillin, and 100 μg/mL streptomycin) at 37 °C under 5% CO2/95% air atmosphere. Twenty-four hours prior to the experiments, the endothelial cell cultures were refreshed with a serum-reduced (10%) medium without crude endothelial cell growth factor. Cell viability, estimated by trypan blue exclusion, was greater than 95% before each experiment.
Human dermal microvascular endothelial cells (abbreviated as HMVEC-D or HMVECs) were purchased from Lonza (Lonza Walkersville, Inc., Walkersville, MD, USA) and cultured according to the supplier’s instructions. Briefly, cells were cultured in EGMTM-2MV (endothelial growth medium), containing basal medium supplemented with human epidermal growth factor (hEGF), vascular endothelial growth factor (VEGF), R3-insulin like growth factor-1 (R3-IGF-1), ascorbic acid, hydrocortisone, human fibroblast growth factor-beta (hFGF-β), fetal bovine serum (FBS), and gentamicin/amphotericin-β (GA) solution (Lonza, USA) at 37 °C under 5% CO2/95% air atmosphere.
Human dermal microvascular endothelial cells (abbreviated as HMVEC-D or HMVECs) were purchased from Lonza (Lonza Walkersville, Inc., Walkersville, MD, USA) and cultured according to the supplier’s instructions. Briefly, cells were cultured in EGMTM-2MV (endothelial growth medium), containing basal medium supplemented with human epidermal growth factor (hEGF), vascular endothelial growth factor (VEGF), R3-insulin like growth factor-1 (R3-IGF-1), ascorbic acid, hydrocortisone, human fibroblast growth factor-beta (hFGF-β), fetal bovine serum (FBS), and gentamicin/amphotericin-β (GA) solution (Lonza, USA) at 37 °C under 5% CO2/95% air atmosphere.