ABSTRACT Inherited retinal degeneration is a class of disease characterized by progressive photoreceptor cell death and irreversible vision loss. While multiple treatment options are being explored, the preferred approach will depend on how far a patient's disease has progressed at the time of diagnosis. For patients who have lost most of their photoreceptor cells and no longer have useful vision, photoreceptor cell replacement therapy will be required. Use of pluripotent patient-derived stem cells to generate transplantable photoreceptor precursor cells is a promising approach that is being aggressively developed. Robust retinal differentiation protocols that enable production of 3D retinal organoids have been reported and adapted to current good manufacturing practices. While retinal organoids contain therapeutically relevant photoreceptor precursor cells, they also contain all the other cell types present in the neural retina. As such, photoreceptor cell enrichment is required in order to achieve optimal treatment outcomes. Photoreceptor cell enrichment has been achieved via the use of fluorescence activated cell sorting and magnetic bead-based pull down, both of which typically rely on the use of cell type specific antibodies that target cell surface antigens. While highly effective, these approaches are challenging to adopt to clinical manufacturing of autologous products. Given that photoreceptor cells naturally segregate to the outermost layer of retinal organoids, we have found that partial organoid dissociation can be used to isolate a relatively pure photoreceptor cell population. While encouraging, we believe that the rate at which retinal organoids dissociate will vary between patients and be highly dependent on organoid shape and size as well as the lot of enzyme being used. For clinical use, the manufacturing process should result in a final product that consistently meets a predetermined set of clinical release criteria with little variability across production runs. As such, standardization of our partial dissociation protocol and validation of the final cell product is essential. To address these needs, in this application we propose two specific aims. In Aim 1, we propose to refine our partial retinal organoid dissociation protocol and identify the optimum tradeoff between photoreceptor cell purity and recovery that is achievable without antibody-based sorting. In doing so, we will fully characterize the identity of all cell types within the final preparation and determine the size and mechanical properties of therapeutic photoreceptor precursor cells. In Aim 2, we propose to fabricate a retinal organoid dissociation and label-free microfluidic photoreceptor cell enrichment platform designed to reduce the amount of time that individual photoreceptor cells spend in dissociation enzyme, enable further photoreceptor cell enrichment, and to permit empirical determination of organoid dissociation state. Specifically, by incorporating...