The outer blood–retinal barrier is composed of a monolayer of retinal pigment epithelium, Bruch's membrane and the choriocapillaris which is fenestrated. Endothelial proliferation and breaching of Bruch's membrane leads to the neovascular form of age-related macula degeneration (ARMD). The aim of this study was to generate an in vitro model that mimics more faithfully the phenotype of the choriocapillaris and the trilayer architecture in vitro. A trilayer culture model was generated with retinal pigment epithelium (ARPE-19) cell cultures on the epithelial surface of amniotic membrane and with human umbilical vein-derived endothelial cells on the other surface. A control model for the effect of retinal pigment epithelium on endothelial changes was generated with corneal epithelial cells replacing the ARPE-19. Both human umbilical vein-derived endothelial and ARPE-19 cells formed confluent monolayers on respective surfaces of the amnion. The human umbilical vein-derived endothelial cells in the trilayer became fenestrated when co-cultured with the ARPE-19 cells, but not with corneal epithelial cells, or when grown as monolayers on the amnion, showing a loss of fidelity of origin in the presence of ARPE-19 cells. These cells also revealed VE-cadherin and ZO-1 at cell–cell contacts from 24 h in the trilayer. The tight junctional molecules, occludin and ZO-1, were localized to cell–cell contact regions in the retinal pigment epithelium, both in the monolayer and in the trilayer system. Permeability of the trilayer was tested by using fluorescein and fluorescein-conjugated tracers under flow. At 72 h the trilayer severely restricted transfer of sodium fluorescein (NaF) (ten-fold reduction) whilst transfer of a 4 kDa FITC-conjugated dextran was virtually occluded, confirming a restrictive barrier. Ultrastructural studies showed the retinal pigment epithelium monolayer was polarized with microvilli present on the apical surface. Paracellular clefts showed numerous tight junctional-like appositions, similar to that seen on amnion alone. This study demonstrates that ARPE-19 and human umbilical vein-derived endothelial cells can be co-cultured on the amniotic membrane and that the resultant cross-talk leads to formation of a fenestrated endothelium, whilst maintaining a polarized restrictive epithelial layer. The fenestrated endothelial phenotype achieved in this human in vitro trilayer model is a first and offers an outer-retinal barrier which approaches the in vivo state and has potential for studies into induced junctional disruption, endothelial proliferation and migration: features of ARMD
To submit an update or takedown request for this paper, please submit an Update/Correction/Removal Request.