Abstract
Purpose :
One of the current limitations of retinal transplantation of stem cells as well as other cell types is the loss of cells into the vitreous chamber and subsequent low survivability. Gelatin-Hydroxyphenylpropionic acid (Gtn-HPA) is a biodegradable hydrogel that can undergo hybridization in vivo, which can result in preservation of injected cells. We tested the hypothesis that Gtn-HPA hydrogel supports survival and integration of RPCs post-transplantation.
Methods :
Human RPCs (hRPCs) and transgenic GFP+ pig RPCs (pRPCs) were maintained in low oxygen conditions as previously published. For in vitro viability studies, 100ul of Gtn-HPA solution was mixed with an equal volume of hRPC cell suspension of various concentrations, to an end concentration of 1-30k cell/ul. Peroxidase and H2O2 at previously published concentrations were added to the mix and spread onto non-treated 6-well plates for gelatin hybridization. Cells in gel were tested for viability using Calcein AM and Ethidium homodimer-1 staining. For in vivo studies, 25 female Long Evans rats immunosuppressed with cyclosporine A were subretinally injected into the left eye with a mixture of GFP+ pRPC, Gtn-HPA:HBSS, peroxidase, and H2O2. Rats were sacrificed at days 7, 10, and 14 for immunostaining of eye cryosections for GFP and DAPI.
Results :
Maximum average viability in vitro of hRPCs in Gtn-HPA was seen with cell concentrations in the 5k-15k/ul range. At 15k/ul, average viability was 71.57±5.9% vs 56.86±2.3% vs 47.53±0.87% at days 1, 4, and 7 (n=3), after start of cell culture in Gtn-HPA. Eyes obtained after in vivo injection of GFP+ pRPCs showed positive GFP on inverted fluorescent microscopy of cryosections, mainly along the inner nuclear layer (Fig 1, 10x). Of the total 13 eyes confirmed to have a subretinal bleb after injection, 3 out of 4 eyes (75%) obtained at day 7, 1 out of 4 eyes (25%) at day 10, and 2 out of 5 eyes (40%) at day 14 showed GFP+ signaling within the retina on consecutive slides.
Conclusions :
Gtn-HPA as an in situ hybridizing hydrogel is able to support hRPC survival in vitro and allows pRPC to survive in the subretinal space in vivo.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.