Tissue Engineered Model of the Outer Neural Retina and Retinal Pigment Epithelium

Abstract

Purpose: Retinal degenerative diseases (RD) that target photoreceptors (PR) or the adjacent retinal pigment epithelium (RPE) affect millions of people worldwide and is characterized by the progressive loss of PR that are largely responsible for vision. The etiology is attributed primarily to RPE cells that exist as a monolayer underlying and supporting the neural retina. Effective treatment for RD have been widely investigated, however there is no in vitro model capable of recreating the cellular organization found in the outer neural retina and its interaction with the RPE. Here we describe an electrospinning (ES) method used to form a fibrous scaffold directly on living RPE cells, designed to direct PR outer segments toward an apical RPE membrane.

Methods: RPE was isolated from adult mouse and human were seeded onto glass coverslips coated with PDL and laminin and cultured to confluence. Polylactic Acid (PLA) was electrospun directly onto samples and RPE cell survival analyzed. Scaffolds were then seeded with a PR enriched retinal suspension and cultured together. Samples were fixed and stained for Bestrophin (RPE) and Recoverin (PR). Cell and scaffolds were imaged by confocal microscopy and environmental scanning electron microscopy (eSEM).

Results: ES PLA fibers formed on living RPE were imaged by both confocal imaging and eSEM showed discrete fibers above living cells. Following 5 days in culture, RPE were able to proliferate to near confluence, but were still restricted to growth beneath the ES scaffold. PR seeded on these scaffolds were observed to form a separate layer with cell bodies found above the fibers and processes extending along fibers towards the RPE layer mimicking the natural organization of the outer retina.

Conclusions: By forming ES scaffolds directly above the RPE we are able to form a surface which restricts the growth of the RPE to a monolayer. This scaffold also provides a support for the seeding of PR with pore sizes small enough to restrict the migration of both RPE and PR cell bodies but large enough to allow the extension of neural processes and outer segments. Using this scaffold we have created a model which recreates the 3D organization of the outer retina, allowing us to study the interaction of these two cells and therapies for the treatments of RD.

Keywords: 694 retinal culture • 648 photoreceptors