Figure 6. Spatially discrete cerebral organoids grow on uncoated scaffolds.

a) Bright-field images show cerebral organoids grown on scaffolds, with procedural control organoids and conventional control organoids shown for reference. On day 9, before Matrigel’s addition, the smooth and optically translucent organoid edges are denoted by the black arrowhead. At day 17, after Matrigel addition and CHIR pulse, neuroepithelial buds are denoted by the black arrowhead. Scale bar: 500 μm. b) Representative tiled overview image of the entire scaffold with organoids at day 20 (N = 3 independent scaffolds). c) Maximum diameter of organoids at day 20. Analysis was performed on organoids using a custom Fiji macro (n = minimum of 5 organoids from 3 independent scaffolds). Data represent mean ±s.d. **** p < 0.0001 and ** p < 0.001, determined by Kruskal–Wallis with Dunn’s post-test. d) Comparison of organoid circularity at day 20 obtained through image analysis using the inbuilt measure function in Fiji (n = minimum of 5 organoids from 3 independent scaffolds). Data represent mean ±s.d. **** p < 0.0001, determined by Kruskal–Wallis with Dunn’s post-test. e) Representative tiled overview image of fixed organoids on 1000 μM spaced scaffolds at day 48. Magnified regions are marked by a red dashed line. Scale bars: 1000 μM (top) and 500 μM (bottom). f) Immunostaining characterization of day 20 cerebral organoids on scaffolds, compared to procedural and conventional control organoids. On the left, histological sections show organoids immunostained with forebrain marker FOXG1 (grey). On the right, histological sections show organoids immunostained with neuronal marker TUJ1 (green), and neural progenitor marker SOX2 (magenta) (n = minimum of 4 organoids). Scale bars: 200 μm. g) Histological sections of day 40 organoids immunostained with dorsal forebrain markers PAX6 (magenta), neuronal marker TUJ1 (green), and FOXG1 (white), Neurons are seen to grow inside the organoid along the scaffold fibers (white arrow). Scale bar: 500 μm. h) Histological sections of day 40 organoids immunostained with ventral marker GSX2 (red) and counterstained with DAPI (blue), and dorsal markers PAX6 (magenta) and TBR1 (green). Scale bar: 100 μm.