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 electrodes into a microfluidic channel, we believe we will be able to monitor
organoid dissociation rate in real time via the Coulter principle. Despite differences that may exist between
patients, rounds of differentiation, or reagents used, we hypothesize that real-time monitoring of dissociation
will allow us to identify when the process should be halted in order to achieve a consistent, high purity product
without the need for further label-based sorting.