Purpose: To establish an ethical, reliable, and expandable retinal pigment epithelial (RPE) cell model with maintained RPE properties compatible with multifarious assays.
Methods: RPE cells from abattoir-obtained porcine eyes were cultured under various conditions. Morphology, RPE cell-specific protein markers (RPE-65, CRALBP), and the tight junction marker ZO-1 were analyzed by phase-contrast microscopy, immunocytochemistry, and western blot, and transepithelial electrical resistance (TEER) was determined to assess barrier function.
Results: The porcine RPE cells (pRPE) were best established using TrypLE Express, 10% fetal bovine serum (FBS) supplemented high-glucose media, and subculturing at semi-confluency. The pRPE cells maintained epithelioid morphology with ZO-1 positive tight junctions at the cell-to-cell borders, the ability to establish proper barrier function (TEERmax: 346/375 Ω⋅cm2 at passage I/passage VI), and expressed CRALBP and RPE-65 for several passages. The RPE characteristics decreased and disappeared with transdifferentiation.
Conclusions: This work describes, for the first time, a pRPE cell model that exhibits preserved RPE properties for several passages on cell culture plastic plates. Though RPE characteristics were maintained for at least 6 passages, the reduced CRALBP and RPE-65 with passaging emphasize that lower passage cells are advantageous to utilize, and that morphology, barrier function, and ZO-1 localization cannot be solely employed as a quality measure of RPE identity. Pigs are phylogenetically similar to humans, including similar physiology, anatomy and immune system. Therefore, porcine RPE cells constitute a relevant model system for studying human eye diseases, such as AMD.
Keywords: 3R; AMD; RPE cell model; RPE-specific protein markers; transepithelial electrical resistance (TEER).