Fig 1. Morphological characteristics of control and RT retinal organoids before and after exposure to RT conditions.

(A) Schematic diagram of RT experiment. (B) Representative examples of hiPSCs-derived retinal organoids at day 360 of differentiation. Top images represent control retinal organoids that remained in a humidified environment at 37°C with 5% CO₂. Bottom images represent retinal organoids that were kept at RT for 5 days. Day -5 represents the first day of storing organoids at RT, day 0 represents the day that the organoids were transferred to the incubator at 37 °C with 5% CO₂, and day 15 represents the recovery period of the organoids, before their collection, in a humidified environment at 37°C with 5% CO₂. Scale bars = 100 μm. (C) The thickness of the neuroepithelial layer across the whole organoid was measured in μm using the ImageJ software. Data are shown as mean ± SEM of 8 representative organoids from each group. An unpaired t-test was performed to estimate differences in the neuroepithelial thickness between the control and RT organoids showing no significant differences between the groups (p = 0.3713). (D) Immunohistochemical analysis of retinal markers of control and RT retinal organoids after 15 days of recovery from storage at RT for 5 days. Expression of photoreceptors (Recoverin, green) and amacrine and ganglion cells (HuC/D, red), Müller cells (Vimentin–green, Sox9 -red), bipolar cells (PKCα, red), Rod photoreceptors (Rhodopsin, green), ganglion cells (SNCG, green), amacrine cells (AP2α, red), and S cone photoreceptors (Opsin SW, green), L/M cone photoreceptors (Opsin MW/LW, green) and horizontal cells (Prox1, green). Nuclei are counterstained with Hoechst (Hoe, blue). Scale bar = 50 μm.