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, 211 (6), 707-16

Establishment of a Human in Vitro Model of the Outer Blood-Retinal Barrier

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Establishment of a Human in Vitro Model of the Outer Blood-Retinal Barrier

R D Hamilton et al. J Anat.

Abstract

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.

Figures

Fig. 1
Fig. 1
Confocal micrographs of monolayers of RPE cells on amnion alone (A–C) and HUVECs on amnion alone (D–F). In the RPE, cell–cell contacts were immunostained for occludin (A) and ZO-1 (B) but were immunonegative for VE-cadherin (C). C also shows propidium iodide staining of nuclei. D shows cytoplasmic location of occludin in HUVECs. Cell–cell contacts did possess ZO-1 (E), and the AJ molecule, VE-cadherin (F). Magnification was the same for all images.
Fig. 3
Fig. 3
Confocal micrographs of trilayers of RPE cells and HUVECs at 72 h co-culture. HUVECs still retained VE-cadherin (A) and ZO-1 (B) at cell–cell contacts. (C) Dual labelling of A and B. (D) Localization of occludin in HUVECs is now predominantly at cell–cell contacts as well as cytoplasm (nuclei stained with PI). (E) Double-immunolabelling of RPE, which is immunonegative for VE-cadherin (green) but positive for ZO-1 (red). Occludin is present at cell–cell contacts (F).
Fig. 2
Fig. 2
Toluidine blue-stained trilayer showing monolayers of the retinal pigment epithelium (RPE) (above) and the human umbilical vein endothelial cells (HUVECs) (below). Note the clear separation of the two monolayers.
Fig. 4
Fig. 4
Confocal micrographs of optical sections of the trilayer tilted around its axis. The images have been tilted at varying angles [starting with the RPE surface (A) to the HUVEC surface (D)] using Velocity software. The trilayer shows immunostaining for ZO-1 (green) and occludin (red) with the yellow showing the areas of dual labelling. Note both HUVECs and RPE cells are present as a continuous monolayer.
Fig. 5
Fig. 5
Electron micrographs of the trilayer. The RPE layer shows a polarized phenotype in possessing microvilli (A) and paracellular clefts between adjacent cells containing junctional regions (B,C). (C) Higher magnification showing that total fusion of membrane does not occur at tight junctional apposition.
Fig. 6
Fig. 6
The endothelial layer of the trilayer shows presence of both paracellular clefts and fenestrae (A); numerous caveoli-like vesicles can be seen, suggestive of fenestra-formation (B,C); paracellular clefts are clearly defined (D,E).
Fig. 7
Fig. 7
Electron micrographs of trilayer where corneal epithelial cells are co-cultured with HUVECs. (A) Both apical microvilli and paracellular cleft are present in HCE-T, whilst the HUVECs remain as a continuous monolayer; no fenestrae can be seen (B).
Fig. 8
Fig. 8
Graph plotting leakage of 4-kd fluorescein dextan tracer across amnion alone, amnion + HUVEC, amnion + RPE, HCE-T + HUVEC co-culture, and the trilayer (RPE + HUVEC co-culture) (*shows sodium fluorescein as a comparison). Severe restriction of tracers can be seen in the trilayer plots.
Fig. 9
Fig. 9
Graph showing levels (pg mL−1) of VEGF produced by the trilayer over 72 h.

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