Isolation of Primary Porcine Retinal Pigment Epithelial Cells for In Vitro Modeling.

The retinal pigment epithelium (RPE) is a crucial monolayer in the outer retina responsible for supporting photoreceptors. RPE degeneration commonly occurs in diseases marked by progressive vision loss, such as age-related macular degeneration (AMD). Research on AMD often relies on human donor eyes or induced pluripotent stem cells (iPSCs) to represent the RPE. However, these RPE sources require extended differentiation periods and substantial expertise for culturing. Additionally, some research institutions, particularly those in rural areas, lack easy access to donor eyes. While a commercially available immortalized RPE cell line (ARPE-19) exists, it lacks essential in vivo RPE features and is not widely accepted in many ophthalmology research publications. There is a pressing need to obtain representative primary RPE cells from a more readily available and cost-effective source. This protocol elucidates the isolation and subculture of primary RPE cells obtained post-mortem from porcine eyes, which can be sourced locally from commercial or academic suppliers. This protocol necessitates common materials typically found in tissue culture labs. The result is a primary, differentiated, and cost-effective alternative to iPSCs, human donor eyes, and ARPE-19 cells.

Utilizing Recombinant Spider Silk Proteins To Develop a Synthetic Bruch's Membrane for Modeling the Retinal Pigment Epithelium.

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.