To date full recapitulation of in-vivo retinal development and structure within hiPSC derived 3-dimensional organoids is limited with respect to both, synchrony of retinal maturation across cell types as well as the characteristic retinal layering. Furthermore, retinal organoids derived from current culture protocols are extremely sensitive to high-throughput handling and require labor-intensive selection procedures to achieve reproducible yields of differentiation, as batch variations in differentiation and maturation remain high. In order to improve the handling of hiPSC derived retinal organoids on a high-throughput scale this proposal envisions the encapsulation of retinal organoids in stiffness tunable hydrogel to enhance resistance to sheer stress during manual/automatic handling. Furthermore, to bridge the discrepancy between cell type specific maturation and lamination observed within current culture systems, it proposes the generation of 3-dimensional organoids from 3D-printed, multilayered spheroids, using differentially aged retinal cell populations. While all neuronal cell types of the retina will be differentiated from hiPSC according to already existing protocols, the here proposed protocol envisions the isolation of each cell type from those “native” organoids, followed by the reassembly into 3D-printed, multilayered spheroids to achieve the characteristic laminar structure of the retina. To enhance cell viability and to mimic in-vivo extracellular matrix conditions each layer of the proposed spheroid will be derived from a stiffness tunable hydrogel matrix, adjusted for each individual cell type. In conclusion, by disentangling the process of cellular differentiation and neuronal network formation, this proposal aims to provide an organoid system that can be generated and maintained within high-throughput setups and provides the ability to simultaneously maintain all neuronal cell types of the retina within an in-vivo representative laminar scaffold.
Solution for: NEI 3-D Retina Organoid Challenge (3-D ROC)
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