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. 2023 Oct 9;11:1251195. doi: 10.3389/fbioe.2023.1251195

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Copyright © 2023 Ko, Spitz, Pramotton, Barr, Xu, Pavlou, Zhang, Tsai, Maaser-Hecker, Jorfi, Choi, Tanzi and Kamm.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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Permeability and Morphology of the BBB Microvascular Network Co-Cultured with the Neurospheres and Aβ Deposition in Microvascular Networks in Co-Culture with ReN-Ctrl and ReN-AD Neurospheres (A) Fluorescence image of the BBB network (red) perfused with 40 kDa fluorescently-labeled dextran (blue). (B) Fluorescence image of a self-assembled network of the human BBB in direct contact with ReN-Ctrl neurospheres (GFP; gray) after 7 days of co-culture. The network was perfused with a 40 kDa fluorescently-labeled dextran (red). (C) Immunofluorescence images of vascular networks after 7 days of co-culture with ReN-Ctrl (top panel) and ReN-AD (bottom panel) neurospheres. Endothelial cells (GFP) are labeled in grey, pericytes (PDGFR) are labeled in magenta, and astrocytes (GFAP) are labeled in cyan. Arrows indicate activated astrocytes in ReN-AD co-cultures. (D) Comparative analysis of morphological parameters extracted from BBB networks after 7 days of co-culture with ReN-Ctrl and ReN-AD neurospheres (n ≥ 4, 3 independent experiments). (E) Left panel: violin plot depicting the permeability values calculated for BBB networks after 7 days of co-culture with ReN-Ctrl neurospheres and ReN-AD neurospheres (n ≥ 4, 3 independent experiments). Right panel: perfused vascular network at timepoint 1 (right after perfusion; top panel) and timepoint 2 (12 min later; bottom panel). ReN-AD neurospheres are indicated as NS; arrows indicate dextran leakage. Overview (F) and representative (G) immunofluorescence images depicting spatially localized 3D6-positive Aβ deposition (Cy5) in microvascular networks of the BBB (GFP) after 7 days of co-culture with ReN-Ctrl and ReN-AD neurospheres. Created with BioRender.com.