Expression of BBB markers, stability, reproducibility and functional properties of a monolayer of human BLECs.

<p>(A) BLECs were obtained by the co-culture of CD34⁺-derived ECs with pericytes for 6 days in a transwell system. (B–C) Paracellular permeability to lucifer yellow of EC monolayers either cultured alone or with pericytes. (D–E) Paracellular permeability in a co-culture of CD34⁺-derived ECs with pericytes at day 6 obtained from (D) different donors and at (E) different laboratories (P = Portugal (LF); F = France (RC); S = Switzerland (BE)). From B to E, results are Mean ± SEM (n≥4). (F) Expression of BBB markers in BLECs as obtained by immunofluorescence. (G) Electron micrographs of ECs co-cultured with pericytes for 6 days (1) or alone (2). (1) Occlusion of the intercellular space between the ECs. WGA-HRP penetrates partially the intracellular cleft (arrow). (2) No occlusion of the intercellular space between the ECs in 84% of the cases. WGA-HRP penetrates from the luminal compartment (asterisk) through the entire intercellular cleft and is deposited in the underlying matrix (arrows). (H) Gene expression of tight junctions and influx transporters in BLECs and CD34⁺-derived ECs. Results are Mean ± SEM (n = 3). (I) Gene expression of efflux transporters and large molecule receptors in BLECS, i.e., CD34⁺-derived ECs co-cultured with pericytes for 6 days. β actin was used as housekeeping gene (J) Expression of P-gp as evaluated by immunofluorescence. In G and K, bar corresponds to 50 µm. *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001.