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PubMed1997

Comparison of tight junction permeability for albumin in iris pigment epithelium and retinal pigment epithelium in vitro.

BACKGROUND: The degenerative retinal diseases are one of the major causes of visual loss in the western world. Although heterologous RPE transplants rescue the photoreceptors in the dystrophic rat model, rejection remains a major limiting factor. Given the common embryonic origin, iris pigment epithelial (IPE) cells might be able to take over the functions of retinal pigment epithelial (RPE) cells, serving as an autologous graft for transplantation and thereby preventing rejection. One of the main functions of RPE cells is the generation of tight junctions which form the outer blood-retinal barrier. In this study we compared the tight junction permeabilities of IPE and RPE cells isolated from Long Evans rats by measuring their albumin clearances. METHODS: IPE and RPE cells were cultured on semipermeable filter supports with and without the addition of 0.02% ethylenediaminetetraacetic acid (EDTA). At selected intervals, the albumin clearances of the IPE and RPE cells were measured spectrophotometrically and compared. The morphology of the cells was compared using electron microscopy and fluorescent labeling. RESULTS: IPE and RPE cells both restricted the passage of albumin in vitro. After the modulation of tight junctions with 0.02% EDTA, the clearance increased in both types of cells in a similar fashion. The morphology of tight junctions was visualized with electron microscopy. CONCLUSION: These results indicate that the functional barrier for macromolecules is similar in IPE and RPE cells in vitro. This raises the possibility that IPE cells would form tight junctions in the subretinal space, thereby substituting for the blood-retinal barrier normally formed by RPE cells.

Actins↗
PubMed1999

Human and porcine anterior lens capsule as support for growing and grafting retinal pigment epithelium and iris pigment epithelium.

PURPOSE: To establish a method for transplantation of cultured monolayers of RPE and IPE into the subretinal space, anterior lens capsule was evaluated for its suitability to serve as growth support and carrier for transplantation procedures. MATERIALS AND METHODS: Twenty-four anterior lens capsules were obtained from porcine eyes. The same number of human lens capsules was obtained during cataract surgery. Six lens capsules of each species were stored at -80 degrees C. Subsequently, the capsules were transferred onto type-I collagen. A second set of six lens capsules was treated identically except for the cryo treatment. A third set of six capsules was initially exposed to 0.05% trypsin for 30 min. Suspended porcine RPE and IPE cells (5 x 10(4) cells/well) were seeded on the top of each capsule. The remaining six lens capsules served as controls and were incubated in uncoated 12-well dishes without undergoing experimental treatment. The cultures were maintained in a water-saturated atmosphere at 37 degrees C with 5% CO(2). Six days later, viability, morphology, and cell density were determined. The capsules covered by a confluent monolayer of cells were transferred into uncoated wells and cultivated for another 10 days. At the end of the experiment, light and phase-contrast microscopy was performed on all capsules. RESULTS: Storage at -80 degrees C and exposure to trypsin resulted in significant reduction of cellular contamination. The highest cell density was found after 5 days when capsules which had undergone cryopreservation or trypsin exposure served as support for RPE and IPE. The pigment cell layer was firmly attached to the capsules and permitted a transfer to other culture flasks without significant cell loss. The IPE cell layer remained confluent after transfer to uncoated culture flasks, while the RPE cell layer ceased to proliferate 10 days after transfer. CONCLUSIONS: Lens capsules may be suitable for growing and supporting monolayers of pigment epithelial cells. Especially IPE cells formed stable monolayers on anterior lens capsules which could be transferred to secondary culture flasks without inflicting damage on the cells.

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