Establishment and characterization of in vitro BBB models. a Monoculture consisting of brain capillary endothelial cells (BCECs, yellow), a non-contact co-culture of BCECs and astrocytes (green) and a triple culture of BCECs, pericytes (purple) and astrocytes. Astrocytes and pericytes were cultured for 21 and 10 days, respectively. The BCECs were cultured for 3 days until 80% confluent. Puromycin was added to the media for the first 2 days. On day −1 the pericytes and/or endothelial cells were passaged to each side of the inserts and left to adhere for 24 h. On day 0, the inserts used for co- and triple culturing were moved to a plate containing astrocytes, and the BCECs were stimulated with hydrocortisone, cAMP and RO. b The cells were identified based on their expression of the tight junction protein ZO1 (BCECs), alpha smooth muscle actin (α-SMA) (pericytes) and glial fibrillary acidic protein (GFAP) (astrocytes). The cell nuclei were counterstained with DAPI (blue). Scale bars 10 µm. c The maximal TEER values were reached on day 2. Co-cultures (red), and triple cultures (green) displayed TEER values of 299 ± 17 and 331 ± 28 Ω cm2 respectively, while the monoculture (blue) only showed a slight increase in TEER (128 ± 9 Ω cm2). Data are presented as means ± SEM (n = 24). Statistical differences were analysed using a 1-way ANOVA with Tukey’s multiple comparisons post hoc test (***p < 0.001). There was no ignificant difference between co- and triple cultures. d The apparent permeability (Papp) of mannitol in cultured BCECs. Data are calculated based on measurements from 18 inserts with TEER values ranging from 72.4 to 321 Ω cm2. The permeability to mannitol decreases as TEER values increase around 150 Ω cm2, which can be obtained by co-culturing the BCECs with pericytes and/or astrocytes.