Innovative techniques for retinal pigment epithelium transplantation

Abstract

Age-related macular degeneration (AMD) occurs due to changes in Bruch’s Membrane (BrM), leading to Retinal Pigment Epithelium (RPE) dysfunction. RPE transplantation has potential as a regenerative strategy but previous attempts have been unsuccessful because BrM replacement has not been taken into consideration. Previous work at the University of Southampton has led to the development of a 60:40 P(MMA-co-PEGMsuccinimidyl carbonate) electrospun fibrous co-polymer scaffold, as a potential artificial BrM. The aim of this project was to characterise and evaluate the co-polymer scaffold, and to develop methods to use it for RPE transplantation. The characteristics of an ideal BrM substitute, as espoused by Binder et al. (2007), were used as benchmarks. RPE transplanted on an artificial BrM is likely to be positioned on the surface of the existing BrM and therefore needs to be more permeable than BrM. Diffusional flux studies showed that, allowing for differences in thickness, the polymer was 48x more permeable than human post-mortem BrM samples. Measurements of transepithelial electrical resistance across monolayers of ARPE-19 cells and primary rabbit RPE cells, cultured either on the co-polymer surface or directly on to tissue culture plates, supported the existence of tight electrical contacts between neighbouring RPE cells. VEGF concentrations were 50% higher in apical vs. basal compartments of culture wells, confirming RPE polarisation. In vitro evaluation of a novel surgical instrument, POLARIS (Polymer And RPE Injector – Southampton), designed to deliver cell-seeded polymer into the subretinal space, could detect no statistical difference in apoptotic cell death between POLARIS (21%) and the unejected controls (21%). ARPE-19 cells showed no increased cytotoxicity from application of a range of ophthalmic viscoelastic devices during cell culture, with baseline cytotoxicity maintained at less than 20%. This supports the potential use of viscoelastic devices within the subretinal space, to prevent bleb collapse during transplantation. Three-port pars plana vitrectomy was performed in 25 rabbits, and creation of a localised retinal detachment allowed the successful delivery of polymer scaffolds into the subretinal space of 11 rabbit eyes. Pre-operative intravitreal saline was found to increase the likelihood of successful surgical induction of posterior vitreous detachment at the time of surgery. The findings of this study show that the 60:40 P(MMA-co-PEGM-succinimidyl carbonate) electrospun copolymer scaffold mimics the desirable properties of an artificial BrM and allows formation of an electrically resistant, polarised RPE monolayer. The polymer has been successfully transplanted into rabbit eyes. These results support use of this copolymer to facilitate RPE transplantation, as a future potential treatment for AMD.

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Identifiers

ORCID for Andrew Lotery: orcid.org/0000-0001-5541-4305

ORCID for Martin Grossel: orcid.org/0000-0001-7469-6854

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