In Situ Cross-linking Hydrogel as a Vehicle for Retinal Progenitor Cell Transplantation

Abstract

One of the current limitations of retinal transplantation of stem cells as well as other cell types is the dispersion of cells from the injection site (including loss of cells into the vitreous chamber) and low survival after transplantation. Gelatin-hydroxyphenyl propionic acid (Gtn-HPA) conjugate is a biodegradable polymer that can undergo covalent cross-linking in situ, allowing for injection of incorporated cells through a small caliber needle followed by gel formation in vivo. We tested the hypothesis that Gtn-HPA hydrogel supports survival and integration of retinal progenitor cells (RPCs) post-transplantation. In vitro compatibility and in vivo graft survival were assessed by mixing an equal volume of Gtn-HPA conjugate and RPC suspension and triggering enzyme-mediated gelation, using minute amounts of horseradish peroxidase and peroxide. Immunocytochemistry showed >80% survival of cells and minimal apoptosis for cells incorporated into Gtn-HPA, equivalent to controls grown on fibronectin-coated flasks. RPCs undergoing mitosis were seen within the three-dimensional Gtn-HPA hydrogel, but the percentage of Ki-67-positive cells was lower compared with the monolayer controls. For in vivo studies, gel–cell mixture or cell suspension in saline was trans-sclerally injected into the left eye of female Long Evans rats immunosuppressed with cyclosporine A. Grafts survived at the 1 week time point of the study, with Gtn-HPA-delivered grafts showing less inflammatory response demonstrated by anti-leukocyte staining. More eyes in the gel–cell mixture group showed surviving cells in the subretinal space compared with saline-delivered controls, while the number of cells surviving per graft was not significantly different between the two groups. This work demonstrates an injectable in situ cross-linking hydrogel as a potential vehicle for stem cell delivery in the retina.