Real-time acquisition of transendothelial electrical resistance in an all-human, in vitro, 3-dimensional, blood–brain barrier model exemplifies tight-junction integrity

The blood–brain barrier (BBB) consists of endothelial cells, astrocytes, and pericytes embedded in basal lamina (BL). Most in vitro models use nonhuman, monolayer cultures for therapeutic-delivery studies, relying on transendothelial electrical resistance (TEER) measurements without other tight-junction (TJ) formation parameters. We aimed to develop reliable, reproducible, in vitro 3-dimensional (3D) models incorporating relevant human, in vivo cell types and BL proteins. The 3D BBB models were constructed with human brain endothelial cells, human astrocytes, and human brain pericytes in mono-, co-, and tricultures. TEER was measured in 3D models using a volt/ohmmeter and cellZscope. Influence of BL proteins—laminin, fibronectin, collagen type IV, agrin, and perlecan—on adhesion and TEER was assessed using an electric cell-substrate impedance–sensing system. TJ protein expression was assessed by Western blotting (WB) and immunocytochemistry (ICC). Perlecan (10 µg/ml) evoked unreportedly high, in vitro TEE...