Hamilton Robin D, Leach Lopa
School of Biomedical Sciences, University of Nottingham, Nottingham, UK.
Methods Mol Biol. 2011;686:401-16. doi: 10.1007/978-1-60761-938-3_20.
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.
外血视网膜屏障由单层视网膜色素上皮（RPE）、布鲁赫膜和有窗孔的脉络膜毛细血管组成。一种在三维结构中包含所有这些层的体外模型比传统的单层培养具有明显优势。这里的细胞，无论是内皮细胞还是上皮细胞，都处于类似于体内的条件下，并且可以参与细胞间和细胞与基质的相互作用。本章描述了如何构建一种人类三层培养模型，该模型将RPE（ARPE - 19）细胞培养在羊膜上皮表面，将人脐静脉来源的内皮细胞（HUVEC）培养在间质表面。该模型在限制小分子（<4 kDa）运输、RPE中富含闭合蛋白的紧密连接以及有窗孔的内皮细胞方面类似于外视网膜屏障。还描述了用于测试所生成的三层结构特性的技术，这些技术包括对紧密连接和黏附连接的结构及分子占据情况进行成像、通过在流动条件下测量荧光素和荧光素偶联示踪剂来估计三层结构的屏障效率、测量分泌的血管内皮生长因子 - A以及超微结构研究，超微结构研究可用于分析RPE中紧密连接的精细结构和内皮窗孔。
