Fig. 1

High-throughput blood–brain barrier organoid formation with microwell hydrogel arrays. a Schematic of microwell plates in 96-well plate format and representative phase contrast image of a GRi3D organoid array within a single well. Scale bar, 1 mm. b Quantification of blood–brain barrier organoid diameter in GRi3D microwell arrays within 24- or 96-well plate compared to organoids grown on agarose substrate. Each dot represents a single organoid, while different colors represent independent experiments. c Representative images of glass-mounted blood–brain barrier organoids labelled with cell type-specific markers showing the spatial distribution of brain endothelial cells (P-gp, magenta), pericytes (NG2, green) and astrocytes (CellTracker Red, cyan) within blood–brain barrier organoids grown in Gri3D arrays. Nuclei are labelled with DAPI (grey). Upper panels show single confocal sections below the spheroid surface. Scale bar, 100 μm. The yellow line shows the position of the orthogonal cross-section shown in the lower panel. Scale bar, 50 μm. Graphs show the mean line profile intensity fluorescence of each cell-type marker within organoids. Distance in μm is measured from left to right across the ROI shown within the dotted box. d Representative fluorescent image of a live/dead staining showing the viability of blood–brain barrier organoids grown on GRi3D arrays. Calcein AM in green labels live cells whereas ethidium homodimer-1 in magenta labels dead cells. Scale bar, 1 mm. Images on the right show a higher magnification of the boxed area. Scale bar 500 μm