The human BBB model has previously been described in detail [57 (link)]. Endothelial cells (ECs) were derived from CD34+ hematopoietic stem cells isolated from human umbilical cord blood according to a previously published method [58 (link)]. Written and informed consent from the donor’s parents was obtained for the collection of umbilical cord blood, in compliance with French legislation. Once isolated from umbilical cord blood, CD34+ cells were differentiated in vitro into endothelial cells (ECs) using endothelial cell growth medium (EGM; Lonza, Walkersville, MD, USA) containing 50 ng/mL vascular endothelial growth factor (PeproTech, Rocky Hill, NJ, USA) and 20% heat-inactivated fetal calf serum (FCS; Sigma Aldrich, St. Louis, MO, USA). After 15 to 20 days, ECs, now visible in culture dish, were then trypsinized and expanded in 0.2% (w/v) gelatin-coated 100 mm Petri dishes (Corning Inc., Corning, NY, USA) in endothelial cell medium (ECM; Sciencell, Carlsbad, CA, USA) supplemented with 5% (v/v) FCS, 50 µg/mL gentamycin (Biochrom AG, Berlin, Germany), and 1 ng/mL basic fibroblast growth factor (Sigma Aldrich).
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