Engineering multicellular organoid for corneal niche modelling and regenerative therapies

Purpose: The corneal niche is composed of various cell types, including limbal stem cells (LSCs), corneal epithelial cells (CECs), and neuronal cells. LSCs are pivotal for corneal epithelial regeneration, CECs maintain the corneal transparency, and neuronal cells contribute to sensory and trophic functions. Despite advances in corneal research, the interplay among these cell types within the corneal niche remains insufficiently understood. This study aims to engineer organoids combining LSCs, CECs, and neuronal cells to create a robust model for studying the corneal niche and to explore their transplantation potential. Methods: LSCs, CECs, and SHSY‐5Y neuronal cells were separately seeded in their growth mediums. After reaching confluency, cells were trypsinized and co‐cultured to form multicellular organoids using the hanging drop technique [1]. Various parameters including cell number, cell‐cell ratio and incubation time were optimized for organoid formation. The organoids were characterized for their cellular composition, structural integrity, and viability using immunohistochemical staining and confocal microscopy. Functional assays, including cell proliferation and differentiation markers, were performed to assess their regenerative potential. Results: To determine the optimal conditions for organoid formation, we experimented with different ratios of LSCs, CECs, and SHSY‐5Y cells, specifically 1:1:1, 2:2:1, 4:2:1, and 8:2:1. The results showed that the engineered organoids formed under all four ratios exhibited a well‐organized structure with high viability for a week. Functional assays including Ki67 and CD44 revealed active proliferation and differentiation, indicating the regenerative potential of the spheroids. Conclusions: This study underscores the potential use of tri‐coculture organoid as a promising therapeutic strategy for limbal stem cells deficiency and a valuable tool for investigating the complex cellular interactions within the corneal environment. References M. Birgul Akolpoglu, Yasemin Inceoglu, Seda Kizilel, An all‐aqueous approach for physical immobilization of PEG‐lipid microgels on organoid surfaces, Colloids and Surfaces B: Biointerfaces, Volume 186, 2020, 110708, ISSN 0927‐7765, https://doi.org/10.1016/j.colsurfb.2019.110708.