Fig. 1: Establishment of a 3D human NVU model for HSE study.

a Schematic diagram of the human neurovascular unit. b Image of a real chip. c 3D schematic diagram showing cell culture on the NVU chip. The yellow area indicates the HBMECs culture compartment of the blood side (1). The dark gray area indicates the porous PCTE membrane between the blood side and brain side (2). The red area indicates the astrocytes/neurons culture compartment of the brain side (3). The blue area indicates the microglia culture compartment of the brain side (4). d 3 days after seeding cells on the chip, confocal micrographs showing the HBMECs on the chip, immunostained for VE-cadherin, ZO-1 and PECAM-1. e Confocal micrographs showing the astrocytes on the chip, immunostained for S100β and GFAP. f Confocal micrographs showing the neurons on the chip, stained for Tuj-1 and MAP2. g A confocal micrograph showing the co-culture of astrocytes (GFAP) and neurons (Tuj-1) in Matrigel. h Confocal micrographs showing the microglia on the chip, immunostained for CD11b and IBA1. i A 3D configuration image showing BBB interface formed by HBMECs (VE-cadherin) and astrocytes (S100β) after co-culture for 3 days. d–i Representative images were from 3 biological replicates. All experiments were repeated at least 3 times. j FITC-dextran permeability assays for BBB formed by HBMECs alone, or HBMECs and astrocytes, after culture for 3 days (n = 6 for biological replicates; n = 3 for technical replicates). Data are presented as the mean ± SD and were analyzed using an unpaired two-sided Student’s t-test.