Journal: Bioactive Materials

Article Title: Micromolded honeycomb scaffold design to support the generation of a bilayered RPE and photoreceptor cell construct

doi: 10.1016/j.bioactmat.2023.07.019

Figure Lengend Snippet: Microscaffolds seeded with RPE and PRs cells. a) High and b) low magnification brightfield images of microscaffolds before and 24 h after being seeded with hPSCs-derived RPE (RPE) cells. Scale bars: a) 100 μm and b) 500 μm. c) Microscaffolds seeded with RPE cells and analyzed after 7 days by immunocytochemistry staining for ZO-1 (green), MITF (red), and DAPI for nuclei (blue). Scale bars, in c and c’ are 100 μm. c’) magnified view of the dashed square showed in c showing ZO-1 immunostaining (upper c’ panel) and a merged image (lower c’ panel). d) Tilted view of a 3D confocal reconstruction showing RPE cells 7 days after seeding on the microscaffold. Scale bar: 100 μm. e-g) Side views of 3D reconstructed images showing the surface of a single honeycomb well covered by a layer of RPE cells 7 days after seeding. Scale bar: 20 μm. h) Violin plots showing the distribution of the number of RPE cells (in blue) or PRs (in red) within individual HC wells 7 days after seeding with 4 × 10 5 RPE cells/transwell or 3.5 × 10 6 PRs/transwell. The box plots in the middle of each violin plot delineate the first and third quartiles of the distribution and the central white dot in each box plot indicates the median value. i) WA09-CRX +/tdTomato hPSC-derived retinal organoid after 120 days of differentiation. Scale bar: 500 μm. j) Dissociated cells from CRX17-tdTomato + retinal organoids. Scale bar: 100 μm. k) High and l) low magnifications of live cell fluorescence microscopy images of a microscaffold seeded with PRs from dissociated CRX +/tdTomato organoids two days after seeding. Scale bars: k) 100 μm and l) 500 μm. m) Maximum intensity projection (Max IP) image of a microscaffold seeded with CRX +/tdTomato PRs cells, collected 7 days after seeding and immunostained with recoverin (RCVN, green) and tdTomato (red). Scale bar: 100 μm. n) Tilted view of a 3D confocal reconstruction showing CRX +/tdTomato PRs 7 days after being seeded on a microscaffold. DAPI was used to counterstain the nuclei (blue). Scale in x-axis: 50 μm; in z-axis; 40 μm; in y-axis: 50 μm.

Article Snippet: D.M.G. and M.J.P. have an ownership interest in Opsis Therapeutics LLC, which has licensed the technology to generate 3D retinal organoids from pluripotent stem cell sources reported in this publication.

Techniques: Derivative Assay, Immunocytochemistry, Staining, Immunostaining, Fluorescence, Microscopy