Figure 1
From: Hydrogel-based milliwell arrays for standardized and scalable retinal organoid cultures
(A) Macroscopic photograph of an array of milli-wells in PEG hydrogel. Scale bar: 200 μm. (B) Orthogonal reconstruction of a confocal z-stack of a single milli-well to verify their geometrical integrity. Scale bar: 200 μm. (C) Schematic representation of our novel approach. (D) Time course of Crx-GFP mESC-derived retinal organoids in microwell arrays after protocol optimization. (i) Micrograph of a single aggregate 20 h after seeding. (ii) Formation of a rigid, bright neuroepithelium surrounding the aggregate at day 3 of culture. (iii) Day 4, optic vesicle (OV) protrusion from specialized area of the neuroepithelium. (iv) Day 6, rare optic cup-like (OC) formation. (v) Day 7, first detection of photoreceptor differentiation revealed by Crx-GFP expression. (vi) Developing retina at 26 days of culture showing GFP-positive photoreceptors. Scale bars: 100 μm. (E) Tile scan of a retinal organoid milli-well arrays at day 14. Scale bar: 1 mm. (F) Quantification of the organoid-forming efficiency, based on the GFP expression, at different days of culture. In D24, ≈93% of the aggregates contained at least one retinal organoid (G) Size quantification of the GFP-positive optic vesicles (OV) at different differentiation stages. (H, I) Gene expression profiles of ROA-organoids for eye field transcription factors and for cone and rod photoreceptor cells at different differentiation times, i.e. day 7 (D7) in grey, day 11 (D11) in blue and day 14 (D14) in green.
