Whole-mount Retinal Organoid Visualization with Cellular Resolution.

Retinal organoids derived from human induced pluripotent stem cells are intricate 3D structures that mimic the human retina, offering a powerful platform for studying retinal development, disease mechanisms, and potential therapeutic strategies. Moreover, as they are derived from patients, they are becoming increasingly popular as they hold great promise as a tool for personalized medicine. Unlike conventional 2D cell cultures, retinal organoids preserve the native 3D architecture of the retina, allowing for a more realistic representation and enabling more physiologically relevant studies. However, their structural complexity, high cellular density, and diverse composition present significant challenges for characterization. To address these challenges and enhance our understanding of retinal organoid maturation while preserving the 3D context, we combined optical clearing methods with immunolabeling to visualize the entire structure of whole-mount organoids with confocal microscopy. For this, we employed a clearing method compatible with low- and high-numerical-aperture objectives, facilitating full-volume imaging and capturing certain regions of interest with cellular resolution. Using this approach, we identified the morphology and distribution in 3D of the three main neuron paths responsible for the visual information transmission: cone and rod photoreceptors, bipolar and ganglion cells. Our findings shed more light on the visualization techniques to address the spatial organization of retinal cells within the organoid.