Abstract
The retinal pigment epithelium (RPE) performs numerous functions that are indispensable for photoreceptor health and vision. This monolayer of cells is also a major site of insult in inherited and age-related macular degenerations. In vitro models of primary RPE such as human fetal and adult RPE cultures have been invaluable for dissecting disease pathways at the cellular and molecular level. However, numerous studies show that it takes over four weeks for human RPE cell monolayers to become fully polarized after plating on semipermeable membrane supports. Poor persistence of transgene expression over this time period critically limits the applicability of human RPE cultures for live imaging studies required to follow dynamic processes like intracellular trafficking and organelle transport that occur over timescales of milliseconds. Here, we provide a detailed three-step protocol for live imaging of polarized primary RPE using high-speed spinning disk confocal microscopy. Step 1: establish porcine RPE monolayers that undergo differentiation within one week after plating on semipermeable membrane supports; step 2: transfect or transduce RPE using either of two different protocols that result in prolonged transgene expression; and step 3: perform multicolor high-speed live imaging of organelle transport in polarized RPE monolayers. Porcine RPE cells and photoreceptor outer segments were isolated from freshly harvested eyes and plated on collagen-coated Transwell® filters to generate polarized monolayers. After seven days, RPE monolayers were highly pigmented, had TER values ≥ 200 Ω.cm2 and cleared outer segments within 5 hours after phagocytosis. These cells expressed RPE65, localized ZO-1 to the tight junction, Na+,K+-ATPase to the apical membrane and acetylated tubulin to the primary cilium. There was an inverse relationship between initial plating density and the time to differentiation. We used nucleofection to express fluorescently tagged genes in RPE cells prior to plating on filters or baculovirus fusion constructs to transfect polarized monolayers. Both these methods resulted in transfection efficiencies over 40% and transgene expression lasted up to 8 days after plating. These filters were imaged by high-speed spinning disk microscopy to follow tubulovesicular trafficking of lysosomes and actin dynamics in the RPE. Four-dimensional image analysis performed using commercially available software was used to analyze live imaging data. In conclusion, this 3-step protocol describes a powerful method to investigate organelle trafficking and function in real time in the RPE that can be used for answering fundamental questions of RPE cell biology and pathobiology.