Immortalized Human Microvascular Endothelial Cells

HMEC-1 cell line was provided by American Type Culture Collection (ATCC^®, CRL-3243^TM). These cells are human microvascular endothelial cells that were immortalized by transfection with simian virus 40. This cell line features a non-pathological phenotype with applications for in vitro toxicological studies [45 (link),46 ]. HMEC-1 cells were cultured in MCDB 131 medium (Gibco, Fisher Scientific, Illkirch, France) supplemented with 10% fetal bovine serum (FBS, Gibco), 10 mM L-glutamine (Gibco), 10 ng/mL recombinant human epidermal growth factor (EGF, Gibco), 10 U/mL penicillin and 10 µg/mL streptomycin (Gibco) and with 1 µg/mL hydrocortisone, obtained from Sigma-Aldrich (Merck, Saint-Quentin-Fallavier, France). Cells were growth in culture flasks purchased from BD Falcon (Corning, NY, USA) and were incubated with 5% CO₂ at 37 °C. At confluency, cells were rinsed with Dulbecco’s phosphate-buffered saline (D-PBS) and cell passage was operated using 0.05% trypsin-EDTA (Gibco). Cells were counted after a dilution in 0.1% trypan blue solution (Sigma-Aldrich). For the experiments, cells were seeded in either 96-well, 6-well microplates (BD Falcon) or 24-well microplates (Seahorse, Agilent Technologies, Santa-Clara, CA, USA).