Culturing Human Endothelial and Vascular Cells

For most in vitro experiments involving ECs, human umbilical vein ECs, i.e., HUVECs (Cell Applications Inc., San Diego, CA, Catalog # 200p-05n and lot # 3,363 and 2,914) between passages 5–8 pooled from multiple donors were used after testing negative for mycoplasma contamination. The cells were cultured in M199 (Sigma M2520) supplemented with 15% FBS (Hyclone, SH30910.02), β-endothelial cell growth factor (Sigma, E1388), and 100 units/ml penicillin and 100 mg/ml streptomycin (Thermo Fisher Scientific). Human VSMC (HVSMC) were purchased from ATCC (Manassas, VA, USA) (PCS-100-012TM) and cultured in M231 medium with smooth muscle growth supplement (Gibco, Waltham, MA, USA) as previously described (31 (link)). All the cells were maintained at 37°C with 5% CO₂ in a hydrated incubator.