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 TEER values (1200 Ω) and the strongest adhesion. 
Coculturing endothelial cells with astrocytes yielded the greatest resistance 
over time. ICC and WB results correlated with resistance levels, with evidence 
of prominent occludin expression in cocultures. BL proteins exerted differential 
effects on TEER, whereas astrocytes in contact yielded higher TEER values and TJ 
expression.-Maherally, Z., Fillmore, H. L., Tan, S. L., Tan, S. F., Jassam, S. 
A., Quack, F. I., Hatherell, K. E., Pilkington, G. J. Real-time acquisition of 
transendothelial electrical resistance in an all-human, in vitro, 3-dimensional, 
blood-brain barrier model exemplifies tight-junction integrity.