Abstract
The outer blood-retinal barrier is composed of a monolayer of retinal pigment epithelium (RPE), Bruch's membrane, and the choriocapillaris, which is fenestrated. An in vitro model that includes all these layers within a 3-D architecture confers a clear advantage over traditional monolayer cultures. Cells here, whether endothelial or epithelial, reside in conditions resembling that in vivo and can participate in cell-cell and cell-matrix cross talk. This chapter describes how a human trilayer culture model was generated with RPE (ARPE-19) cells cultured on the epithelial surface of amniotic membrane and with human umbilical vein derived endothelial cells (HUVEC) on the interstitial surface. This model resembles the outer retinal barrier both in restricting transport of small molecules (<4 kDa), possession of occludin-rich tight junctions in the RPE and fenestrated endothelial cells. Techniques used to test the generated trilayer properties are also described and these include imaging of structure and molecular occupancy of tight and adherens junctions, estimation of the barrier efficiency of trilayer by measurement of fluorescein and fluorescein-conjugated tracers under flow, measurement of secreted vascular endothelial growth factor-A and ultrastructural studies, which allow analyses of the fine structure of the tight junctions in the RPE, and the endothelial fenestra.