Cell culture and construction of BBB model

Rat brain endothelial cells (RBECs), astrocytes and pericytes were isolated and the in vitro BBB model was constructed with these three cell types, as previously described [18, 19]. The co-cultured cells were maintained in DMEM/F12 supplemented with 10% plasma-derived serum (Animal Technologies), 1.5 ng/ml basic fibroblast growth factor (Roche Applied Sciences), 100 μg/ml heparin, 5 μg/ml insulin, 5 μg/ml transferrin, 5 ng/ml sodium selenite, 50 μg/ml gentamycin and 500 nM hydrocortisone (all from Sigma-Aldrich) and used for experiments 3–5 days after culture.