Fig. 1.

Schematic illustration of the experimental workflow. hCMEC/D3 cells stably expressing Cldn5-YFP were generated by lentiviral transduction. HEK293T cells were used as packing cell line to produce lentiviral particles for transduction of hCMEC/D3-WT cells. At day 0, prior to experiments, pBCECs were freshly isolated from porcine brain. On the same day, pBCECs, hCMEC/D3-WT and -Cldn5-YFP cells were seeded on semipermeable membranes of two chamber devices, each in triplicates. During experiments and cultivation, medium of hCMEC/D3-Cldn5-YFP cells was supplemented with doxycycline (1 µg/mL). After seeding cells attach, proliferate and differentiate to form a continuous monolayer with tight junctions sealing the paracellular cleft between adjacent cells. Transendothelial resistance (TEER) was measured once daily until 7 days after seeding of the cells, using an EVOM Volt-Ohm meter equipped with a STX2/chopstick, consisting of a fixed pair of electrodes. For comparison, TEER of hCMEC/D3 cells was also measured using an EndOhm-6 chamber (not illustrated). At day 7 after seeding, the cells were used for measuring paracellular permeability of mannitol and transcellular drug transport using the Pgp substrate N-desmethyl-loperamide (dLop). Pgp functionality was assessed by rhodamine 123 (Rho123) uptake assay. Another set of cells was grown to analyze Cldn5 and Pgp expression by Western blot (WB) and Cldn5 localization by immunofluorescent staining (IF) 7 days after seeding. Cldn5 expression in hCMEC/D3-Cldn5-YFP cells was also analyzed 21 days after seeding. Drawings are not to scale