citations

                    Modeling of Blood–Brain Barrier (BBB) Dysfunction and Immune Cell Migration Using Human BBB-on-a-Chip for Drug Discovery Research
                

Authors:

Masato Ohbuchi , Mayu Shibuta , Kazuhiro Tetsuka , Haruna Sasaki-Iwaoka , Masayo Oishi , Fumitaka Shimizu, Yasuhisa Nagasaka

In:

Source: Int J Mol Sci
Publication Date: (2024)
Issue: 25: 12

Research Area:

Stem Cells

Drug Discovery

Cells used in publication:

PBMC, human: Species: human; Tissue Origin: blood
CD4+, human: Species: human; Tissue Origin: blood

Culture Media:

Endothelial Cell Growth Medium 2

Experiment

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4.1. Cell Culture

Cell lines of immortalized human brain microvascular endothelial cells (TY10) [30,31], immortalized human brain pericytes (hBPCT) [56], and immortalized human astrocytes (hAST) [57] were provided by Yamaguchi University, Japan. Cells were cultured at 33 °C, 5% CO₂ to allow optimal cell expansion in a collagen type I-coated 60 mm dish (356401, Corning, NY, USA) or 100 mm dish (4020-010, Iwaki, AGC Techno Glass Co. Ltd., Yoshida, Japan). Cell lines were immortalized with an hTERT and a temperature-sensitive SV40 large-T antigen, which allowed the cells to grow at the permissive temperature of 33 °C. TY10 cells were cultured in EGM-2 (CC-3162, LONZA, Basel, Switzerland), which was supplemented with 20% fetal bovine serum (FBS). hBPCT cells were cultured in pericyte medium (#1201, ScienCell, Carlsbad, CA, USA). hAST cells were cultured in astrocyte medium (AM, #1801, ScienCell).

Abstract

Blood–brain barrier (BBB) dysfunction is a key feature in neuroimmunological and neurodegenerative diseases. In this study, we developed a microfluidic human BBB-on-a-chip to model barrier dysfunction and immune cell migration using immortalized TY10 brain endothelial cells, pericytes, and astrocytes. It was found that immortalized TY10 brain endothelial cells developed a microvascular structure under flow. Pericytes were localized on the basal side surrounding the TY10 microvascular structure, showing an in vivo-like structure. Barrier integrity increased under co-culture with pericytes. In addition, both ethylenediaminetetraacetic acid (EDTA) and anti-Claudin-5 (CLDN5) neutralizing antibody caused a decrease in the transendothelial electrical resistance (TEER). EDTA caused the leakage of 20 kDa dextran, suggesting different effects on the BBB based on the mechanism of action, whereas anti-CLDN5 antibody did not cause leakage. In the tri-culture model, human T cells migrated through endothelial vessels towards basal C-X-C motif chemokine ligand 12 (CXCL12). The live-imaging analysis confirmed the extravasation of fluorescence-labelled T cells in a CXCL12-concentration- and time-dependent manner. Our BBB model had an in vivo-like structure and successfully represented barrier dysfunction and transendothelial T cell migration. In addition, our study suggests that the inhibition of CLDN5 attenuates the BBB in humans. This platform has various potential uses in relation to the BBB in both drug discovery research and in elucidating the mechanisms of central nervous system diseases.

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