Characterisation of an In Vitro Model for Retinal Pigment Epithelium - Choriocapillaris Complex

Abstract

Abstract: : Purpose: Further understanding of the pathological processes of Age Related Macular Degeneration (ARMD) is paramount for future treatment regimes of the disease. The purpose of this study was to produce an optimum model for the RPE - Bruch's membrane – choriocapillaris for further investigation. We mimicked the RPE - Bruch's membrane – choriocapillaris complex with Human Umbilical Vein Endothelial (HUVEC) replacing the choriocapillaris and Amniotic membrane instead of Bruch's membrane. Methods: Human Amniotic membrane from normal pregnancies delivered by caesarean section and HUVEC from the umbilical cord from the same section were isolated (N=3). The amniotic membrane was treated with Thermolysin to remove the native epithelial cells. RPE cells from a spontaneously immortalised commercially available source (ARPE-19, ATCC) were cultured in uncoated flasks until confluent and then plated onto the amniotic membrane basement membrane surface at 10-30x10⁴/cm². 24 hrs later, the amniotic membrane was turned over and the HUVEC cells, cultured on gelatin-coated flasks until passage 2 in standard culture medium, were plated onto the interstitial surface at 10-30x10⁴/cm² of the membrane (N=8 for each cord). Permeability studies were performed on amniotic membrane alone, each monolayer cultured on the membrane and on the trilayer. Results: The HUVEC and RPE were confluent on the membrane by 48 hrs as seen by conventional microscopy and remained confluent monolayers for 7 days. The HUVEC and RPE were examined daily with immunocytochemistry of the Adherens and Tight junctions, in particular VE-Cadherin and ZO-1. Confocal microscopy shows the trilayer adherens and tight junctions. Permeability studies of this trilayer confirms tight junctions with 4Kd protein passage being undetected. Conclusions: This model for the RPE - Bruch's membrane – choriocapillaris complex permits exposure to pathological conditions with a view to mimicking early stages of ARMD.

Keywords: age-related macular degeneration • vascular cells • cell adhesions/cell junctions