Characteristics Of Rat Iris Pigment Epithelial Cells Cultured On Modified Expanded-polytetrafluroethylene (ePTFE) Substrates

Abstract

Purpose: : Age-related macular degeneration (AMD) is the leading cause of severe and permanent visual loss in patients above 55 years of age with no effective treatments. The key factors that cause loss of vision are disorders that occurred within the retinal pigment epithelium (RPE) and its overlying photoreceptors. Transplantation of healthy RPE cells into the subretinal space may help rescue the photoreceptors and in turn treating AMD. However, harvesting RPE cells can be a technically complicated procedure. Iris pigment epithelial (IPE) cells, which possess identical embryonic origin and similar properties as RPE cells, are easy to obtain. We aimed to investigate the characteristics of IPE cells grown on modified ePTFE substrates for subretinal transplantation purposes.

Methods: : IPE cells isolated from 3 to 4-week-old Dark Agouti rat eyes were seeded onto different substrates, including fibronectin (5.31µg/ml) n-heptylamine (HA) ePTFE substrate, HA ePTFE substrate and ePTFE substrate with fibronectin (5.31µg/ml) tissue culture polystyrene (TCPS) as control. Cell number was quantified by nuclear counts at day 1, 2, 4, 7, 14 and 28. The formation of tight junction was identified by immunostaining of junction proteins, transmission electron microscopy (TEM), and transepithelial electrical resistance (TEER) value. The capacity of phagocytosis was evaluated by immunostaining of rhodopsin.

Results: : For the IPE cells isolated from Dark Agouti rats, increased cell number was observed in cells grown on fibronectin HA ePTFE substrate and fibronectin TCPS, exhibiting heavy pigmentation and epithelial morphology, while only few cells attached onto HA ePTFE substrate and ePTFE substrate. At Day 28, tight junction formation was indicated by cell-cell junctional proteins along cell borders and increase of TEER value. At electron microscopic level, IPE cells cultured on fibronectin HA ePTFE substrate formed a monolayer with tight junction between two adjacent cells. These cells also demonstrated the ability to phagocytose photoreceptor outer segments in a time-dependent manner.

Conclusions: : IPE cells from Dark Agouti rats cultured on fibronectin HA ePTFE substrate could differentiate and form a monolayer that may be suitable for transplantation.

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