Cultivation of Adult Human Brain Endothelial Cells

Non-immortalized HBMEC originating from adult human brain cortex (Cell Systems, Kirkland, WA, USA, ACBRI 376) were cultivated in gelatin (Serva Electrophoresis, Heidelberg, Germany) coated T-75 culture flasks (Greiner Bio-One, Frickenhausen, Germany). Cells were propagated in RPMI-1640 medium (Sigma-Aldrich), supplemented with 10% fetal calf serum (FCS) (Thermo Fisher Scientific, Waltham, MA, USA), 10% Nu-Serum (BD Biosciences, San Jose, CA, USA), 2 mM L-glutamine (Thermo Fisher), 1 mM sodium pyruvate (Thermo Fisher), 1% minimum essential medium non-essential amino acids (Thermo Fisher), 5 U/ml heparin (Biochrom, Berlin, Germany), and 0.3% endothelial cell growth supplement (Cell Systems). Cultures were kept in a humid atmosphere at 37 °C with 5% CO₂. Confluent monolayers were expanded as described previously [19 (link)], and experiments were coherently conducted with recently thawed cells at passage 8. Basic endothelial cell attributes of HBMEC (characteristic spindle-shaped growth pattern and expression of the endothelial marker CD31) as well as inducibility of intercellular adhesion molecule 1 had been confirmed in preliminary experiments [19 (link)].