Quantitative Analysis of Possible Surgical Shear Force Stress on Polymer Substrates Seeded With Human Embryonic Stem Cell (hESC) Derived Retinal Pigment Epithelium (RPE) Monolayers Implanted into the Rat Subretinal Space

Abstract

Purpose: : Human embryonic stem cell (hESC) derived retinal pigment epithelium (RPE) cells can be cultured as a monolayer on parylene substrates for implantation into the subretinal space of a diseased eye. This study evaluates whether surgical shear force during implantation can affect the preservation of the RPE monolayers by quantification of the number of cells present on the substrate before and after implantation in the subretinal space of an animal model of RPE dysfunction, The Royal College of Surgeons (RCS) rat.

Methods: : hESC (H 9) derived RPE cells are cultured as a monolayer on 10 µm thick parylene substrates divided into rectangular pieces (0.4 mm x 0.9 mm). 28 day old dystrophic RCS rat pups (n=10) were used for subretinal implantation studies (one implant/animal). Total number of cells present on the substrate prior to implantation was calculated from photomicrographs. One day after implantation, animals were sacrificed and the eyes were subjected to histological examination using hematoxylin & eosin staining. High resolution images of retinal sections passing through the substrate were captured using an Aperio scanscope; the total number of cells present on each substrate was estimated using the associated, commercially available software.

Results: : The mean number of cells present on each piece of substrate prior to implantation was 2242±51 (SD, range 2176-2331). Following implantation, in 80% of the rats (8/10), the degree of preservation of the RPE cells on the substrate was above 90% of the pre-implantation level. Major cell loss associated with surgical implantation was noticed only in one rat. Partial disruption of the RPE monolayer structure was observed at certain locations in few other rats.

Conclusions: : hESC-RPE monolayers cultured on parylene substrates can be successfully implanted into the subretinal space of RCS rats without major cell loss due to surgical shear force effects. The results support the use of such implants for therapeutic approaches aimed at slowing the progression of outer retinal dystrophies such as age-related macular degeneration (AMD).

Keywords: retinal pigment epithelium • transplantation • age-related macular degeneration