Cultivation of Adult HBMEC Cells

Non-immortalized adult HBMEC (ACBRI 376, Cell Systems, Kirkland, WA, USA) were grown to confluence in gelatin (Serva Electrophoresis, Heidelberg, Germany) coated T-75 culture flasks (Greiner Bio-One, Frickenhausen, Germany) in a humid atmosphere at 37 °C with 5% CO₂. Cells were propagated in RPMI-1640 medium (Sigma-Aldrich) supplemented with 10% fetal calf serum (Thermo Fisher Scientific, Waltham, MA, USA), 10% Nu-Serum (BD Biosciences, San Jose, CA, USA), 2 mM L-glutamine, 1 mM sodium pyruvate, 1% minimum essential medium with non-essential amino acids (all: Thermo Fisher), 5 U/mL heparin (Biochrom, Berlin, Germany), and 0.3% endothelial cell growth supplement (Cell Systems). As described previously [26 (link)], confluent monolayers were expanded, and recently thawed cells of passage 8 were used for all experiments. Previous experiments had confirmed basic endothelial characteristics [26 (link)].