An easy-to-perform method for microvessel isolation and primary brain endothelial cell culture to study Alzheimer's disease.

Chen, Y; Huang, X; Chen, H; Yi, C

An easy-to-perform method for microvessel isolation and primary brain endothelial cell culture to study Alzheimer's disease.

Chen, Y Huang, X Chen, H Yi, C

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Publisher:: CELL PRESS
Publication Type:: Journal Article
Citation:: Heliyon, 2024, 10, (12), pp. e33077
Issue Date:: 2024-06-30

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Field	Value	Language
dc.contributor.author	Chen, Y
dc.contributor.author	Huang, X
dc.contributor.author	Chen, H
dc.contributor.author	Yi, C
dc.date.accessioned	2024-08-06T01:21:52Z
dc.date.available	2024-06-13
dc.date.available	2024-08-06T01:21:52Z
dc.date.issued	2024-06-30
dc.identifier.citation	Heliyon, 2024, 10, (12), pp. e33077
dc.identifier.issn	2405-8440
dc.identifier.issn	2405-8440
dc.identifier.uri	http://hdl.handle.net/10453/180032
dc.description.abstract	Dysfunction of the blood-brain barrier (BBB) has been increasingly recognised as a critical early event in Alzheimer's disease (AD) pathophysiology. Central to this mechanism is the impaired function of brain endothelial cells (BECs), the primary structural constituents of the BBB, the study of which is imperative for understanding AD pathophysiology. However, the published methods to isolate BECs are time-consuming and have a low success rate. Here, we developed a rapid and streamlined protocol for BEC isolation without using transgenic reporters, flow cytometry, and magnetic beads, which are essential for existing methods. Using this novel protocol, we isolated high-purity BECs from cell clusters of cortical microvessels from wild-type and APPswe/PS1dE9 (APP/PS1, a classical AD model) mice at 2, 4 and 9 months of age. Reduced levels of tight junction proteins Claudin-5 and Zonula Occludens-1, as well as glucose transporter 1, were observed in the isolated cortical microvessels from APP/PS1 mice and amyloid-β (Aβ) oligomer-treated BECs from wild-type mice. Trans-well permeability assay showed increased FITC-dextran leakage in BECs treated with Aβ, suggesting impaired BBB permeability. BECs obtained using our novel protocol can undergo various experimental analyses, including immunofluorescence staining, western blotting, real-time PCR, and trans-well permeability assay. In conclusion, our novel protocol represents a reliable and valuable tool for in vitro modelling BBB to study AD-related mechanisms and develop targeted therapeutic strategies.
dc.format	Electronic-eCollection
dc.language	eng
dc.publisher	CELL PRESS
dc.relation.ispartof	Heliyon
dc.relation.isbasedon	10.1016/j.heliyon.2024.e33077
dc.rights	info:eu-repo/semantics/openAccess
dc.title	An easy-to-perform method for microvessel isolation and primary brain endothelial cell culture to study Alzheimer's disease.
dc.type	Journal Article
utslib.citation.volume	10
utslib.location.activity	England
pubs.organisational-group	University of Technology Sydney
pubs.organisational-group	University of Technology Sydney/Faculty of Science
pubs.organisational-group	University of Technology Sydney/Strength - CHT - Health Technologies
pubs.organisational-group	University of Technology Sydney/Faculty of Science/School of Life Sciences
utslib.copyright.status	open_access	*
dc.rights.license	This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License (CC BY-NC 4.0). To view a copy of this license, visit https://creativecommons.org/licenses/by-nc/4.0/
dc.date.updated	2024-08-06T01:21:44Z
pubs.issue	12
pubs.publication-status	Published online
pubs.volume	10
utslib.citation.issue	12

Abstract:

Dysfunction of the blood-brain barrier (BBB) has been increasingly recognised as a critical early event in Alzheimer's disease (AD) pathophysiology. Central to this mechanism is the impaired function of brain endothelial cells (BECs), the primary structural constituents of the BBB, the study of which is imperative for understanding AD pathophysiology. However, the published methods to isolate BECs are time-consuming and have a low success rate. Here, we developed a rapid and streamlined protocol for BEC isolation without using transgenic reporters, flow cytometry, and magnetic beads, which are essential for existing methods. Using this novel protocol, we isolated high-purity BECs from cell clusters of cortical microvessels from wild-type and APPswe/PS1dE9 (APP/PS1, a classical AD model) mice at 2, 4 and 9 months of age. Reduced levels of tight junction proteins Claudin-5 and Zonula Occludens-1, as well as glucose transporter 1, were observed in the isolated cortical microvessels from APP/PS1 mice and amyloid-β (Aβ) oligomer-treated BECs from wild-type mice. Trans-well permeability assay showed increased FITC-dextran leakage in BECs treated with Aβ, suggesting impaired BBB permeability. BECs obtained using our novel protocol can undergo various experimental analyses, including immunofluorescence staining, western blotting, real-time PCR, and trans-well permeability assay. In conclusion, our novel protocol represents a reliable and valuable tool for in vitro modelling BBB to study AD-related mechanisms and develop targeted therapeutic strategies.

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http://hdl.handle.net/10453/180032