PURPOSE: The retinal pigment epithelium (RPE) plays an important role in the pathogenesis of age-related macular degeneration (AMD) and other retinal degenerative diseases. The introduction of healthy RPE cell cultures into the subretinal space offers a potential treatment strategy. The aim of this study was the long-term culture and characterisation of RPE cells on nanofiber scaffolds. METHODS: Nanofiber scaffolds consisting of polycaprolactone (PCL) and collagen were prepared by electrospinning. Porcine RPE cell cultures were maintained on PCL scaffolds, PCL-collagen scaffolds, and controls at the bottom of 24-well plates. Cell culture analysis was performed by immunohistochemistry, while the release of inflammatory cytokines IL-6, IL-8, TNF-α, and PDGF-β was measured by ELISA and multiplex assays. Ultrastructural features were examined by transmission electron microscopy. RESULTS: The observation period averaged 42.7 weeks for controls, 38.7 weeks for PCL scaffold cultures, and 36.1 weeks for PCL-collagen scaffold cultures, with cell number and morphology remaining stable. TNF-α levels in the supernatants were minimal, IL-6 levels were consistently low, and IL-8 levels decreased from initially high to lower levels over time. CONCLUSION: RPE cells were stably cultured on nanofiber scaffolds for extended periods of time. The long-term physiological properties of RPE cells, including phagocytic ability and visual cycle enzyme activity, need to be further investigated before clinical application. In addition, controlling the expression of inflammatory mediators is a major challenge. Despite these hurdles, overcoming them is critical given the increasing prevalence of retinal degenerative diseases.