Fig 3. Evaluation of the blood-brain barrier integrity.

(A) The transendothelial electrical resistance (TEER) (Ω*cm²) of mouse, rat, and porcine brain capillary endothelial cells (mBECs, rBECs, pBECs) cultured in mono-culture (black triangles) and co-culture with astrocytes (blue circles). Co-culturing the BECs with astrocytes causes an increase in TEER compared to mono-cultured BECs. Each triangle or circle represents an individual culture insert showing a great variation in TEER across the inserts with identical culture conditions. Both mBECs and rBECs show a rapid increase in TEER after induction (day 1), peaking around day two to three, oppositely to pBECs, which have a slower increase in TEER peaking around day four. The data are illustrated as mean ± standard deviation (SD) (mBECs n = 15, rBECs n = 10–11, and pBECs n = 15–18). (B) The functional barrier integrity of the BECs was additionally evaluated by calculating the apparent permeability (Papp) of [3H]-D-Mannitol plotted against TEER across mBECs (black), rBECs (orange), and pBECs (blue) cultured in mono-culture (triangles) or co-culture (circles). The higher the TEER, the lower the Papp; however, in the TEER range of 150–650 Ω*cm² only small differences in the Papp are observed. mBECs n = 12, rBECs n = 12, and pBECs n = 12–16 for each culturing condition. (C) Quantification of cell area of mBECs, rBECs, and pBECs cultured in mono- and in co-culture. pBECs are significantly larger than both mBECs (p = **) and rBECs (p = ***), and co-culturing the BECs does not affect the cell size. The bottom panel shows representative images of how the area of the cells was quantified. Scalebar = 20μM. To analyze the difference in size between mono- and co-cultured BECs within the same species and to compare differences in size between species, a two-way ANOVA with Tukey’s multiple comparisons post hoc test was used. ** p< 0.01, *** p< 0.001. Data are depicted as mean cell area μm² ± SD (n = 4).