isolation-primary-porcine-retinal-pigment-epithelial-cells-for-vitro

Isolation of Primary Porcine Retinal Pigment Epithelial Cells for <em>In Vitro</em> Modeling

The retinal pigment epithelium (RPE) is a crucial monolayer in the outer retina responsible for supporting photoreceptors. RPE degeneration commonly ...

Utah State University

Spider silks are intriguing biomaterials that have a high potential as innovative biomedical processes and devices. The intent of this study was to evaluate the capacity of recombinant spider silk proteins (rSSps) as a synthetic Bruch's membrane. Nonporous silk membranes were prepared with comparable thicknesses (<10 μm) to that of native Bruch's membrane. Biomechanical characterization was performed prior to seeding cells. The ability of RPE cells (ARPE-19) to attach and grow on the membranes was then evaluated with bright-field and electron microscopy, intracellular DNA quantification, and immunocytochemical staining (ZO-1 and F-actin). Controls were cultured on permeable Transwell support membranes and characterized with the same methods. A size-dependent permeability assay, using FITC-dextran, was used to determine cell-membrane barrier function. Compared to Transwell controls, RPE cells cultured on rSSps membranes developed more native-like "cobblestone" morphologies, exhibited higher intracellular DNA content, and expressed key organizational proteins more consistently. Comparisons of the membranes to native structures revealed that the silk membranes exhibited equivalent thicknesses, biomechanical properties, and barrier functions. These findings support the use of recombinant spider silk proteins to model Bruch's membrane and develop more biomimetic retinal models.

Chase A. Paterson

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