Objective: Age-related macular degeneration (AMD) is the leading cause of visual impairment in older adults. Despite extensive studies on AMD, the mechanism of drusen formation remains unknown, limiting the development of therapeutic approaches. Therefore, our goal was to establish a reproducible primary porcine retinal pigment epithelium (RPE) model featuring dry AMD and characterize subsequent RPE morphological changes, the oxidative stress response, and the transcriptome profile at different stages of drusen formation. Methods: Primary porcine RPE cells cultured on permeable Transwell and impermeable plastic surfaces were compared. Barrier functions were assessed via transepithelial electrical resistance measurement and immunostaining. Hydroxyapatite (HAP) deposition was assessed via Alizarin Red S staining and scanning electron microscopy with a focused ion beam. Extracellular glutathione was quantified via Ellman’s reagent. RT‒qPCR and RNA sequencing were used to analyze transcriptomic changes at different stages of drusen progression. Results: In primary porcine RPE cells cultured on plastic dishes, initial ~2 µm HAP-containing spherules were detected after 2 weeks that progressed into large ~30 µm drusen by week 8. Robust deposit accumulation is accompanied by oxidative stress, RPE hyperpigmentation, and apoptosis. RNA-seq analysis revealed gradual upregulation of key epithelial‒mesenchymal transition (EMT) markers (FN1, VIM, and ACTA2) and AMD-related genes (C3, CFH, VEGFA, CLU, and TIMP3) over the 8-week culture period. Conclusion: We established a highly reproducible primary porcine RPE model of dry AMD. Our RPE model enables the investigation of drusen formation and the RPE response across different AMD stages and provides a valuable platform for developing and testing novel therapeutic approaches.
Nemesh et al. (Thu,) studied this question.