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

Abstract

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.

Quantitatively relating brain endothelial cell–cell junction phenotype to global and local barrier properties under varied culture conditions via the Junction Analyzer Program

“…Despite the different properties of the six difference matrices studied here, [13,43] use of different substrate coatings had almost no effect on cell shape factors and had the strongest influence on continuous (and in some cases, punctate) ZO-1 and VE-cadherin junctions. CN, Fbn, F:C:L, and HA/Gtn, all induced similar levels of total junction coverage, in line with previous reports for various brain EC types [14,15,44], while CIV and LN induced less junction localization in some cases.…”

“…Another biomimetic approach has been the use of different matrix proteins that (at least partially) recapitulate the in vivo basement membrane or the brain microenvironment. The basement membrane is known to have an important role in maintaining vascular function [13]. As such, is it unsurprising that constituents of this matrix (i.e., fibronectin, collagen type IV, and laminin; or combinations of the three) are reported to elevate TEER values relative to type I collagen in porcine brain capillary ECs, [14] and promote adhesion and spreading of iPSC-derived brain ECs [15].…”

Loud music and the specific sound stress open the blood-brain barrier: new fundamental, biomedical, and social aspects

“…To answer the question, whether sound opens BBB directly or via stress, we further analyzed BBB permeability to the EBD-albumin complex in anesthetized mice to prevent influences of stress stimuli. Additionally, we studied sound effects on the BBB integrity in in vitro experiments using measurement of trans-endothelial electrical resistance (TEER) 21 .…”

A human co‐culture cell model incorporating microglia supports glioblastoma growth and migration, and confers resistance to cytotoxics

“…20,21 The soluble signals and differentiation factors in the serum-supplemented media also influence cell growth and interaction with exogenous factors. Since within our laboratories we have considerable experience of 3D human cell in vitro modeling in the context of GBM invasion, 24 blood-brain barrier mediated drug delivery, 25 and cancer metastasis, 26,27 we decided to begin a multistage program of developing such 3D models for preclinical drug testing of GBM using all human, multicellular, in vitro conditions. In our laboratories, human serum supplementation has also been shown to modulate both protein and gene expression in human biopsy-derived GBM cells as well as to influence cell shape and proliferation.…”