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. 2023 Feb 10;6:164. doi: 10.1038/s42003-023-04543-5

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© The Author(s) 2023

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 4 — a Screening for the non-freezing preservation conditions. +++: multilayered continuous retinal epithelium was preserved. ++: multilayered continuous retinal epithelium was preserved but neural rosettes appeared. +: multilayered continuous retinal epithelium was observed but very limited. –, no multilayered continuous retinal epithelium was observed after the preservation. b Scheme of the preservation experiments. c Proportions of Crx⁺ photoreceptor precursors in multilayered retinal tissues. The proportions were calculated by dividing the number of Crx⁺ cells by the number of nuclei (DAPI). Data are presented as mean ± SE for n = 5 (4 °C), 4 (12 °C), 4 (15 °C), 5 (17 °C), 5 (20 °C), 5 (22 °C), 4 (37 °C), and 3 (control culture). d Cell viability in non-preserved retinas (Control) and retinas preserved for 4 days. Data are shown as mean ± SE (n = 5 per group). e Gene expressions of Chx10 and Crx in non-preserved retinas (Control) and retinas preserved for 4 days. Data are presented as mean ± SE (n = 3 per group). f IHC analysis of retinas that were not preserved (d70 + 0), preserved in Optisol for 2 days (d70 + 2) and 4 days (d70 + 4), and preserved in Optisol for 4 days followed by recovery culture for 3 days (d70 + 4 + 3) and 7 days (d70 + 4 + 7). Ki67 (red) staining in the upper panels. EdU staining (green) in lower panels. DAPI is shown in blue. Scale bar: 50 µm. g Numbers of EdU-positive cells per 100 µm-wide multilayered retinal tissue in each group. Data are presented as mean ± SE (n = 6 per group). ***p < 0.001 for one-way ANOVA followed by a Tukey’s test. h Scheme for dissection, ring-PCR test, and shipping of retinal sheets. n.s., not significant.