Project Summary/Abstract Age-related macular degeneration (AMD) is a leading cause of visual impairment and blindness in adults over the age of 65 and is expected to affect ~288 million people worldwide by the year 2040. Recently, induced pluripotent stem cells (iPSC)-derived RPE generated from AMD patients and those with phenotypically similar monogenic diseases have been shown to approximate elements of AMD disease phenotype in culture, including the formation of sub-RPE deposits resembling drusen, dysregulated complement, and mitochondrial dysfunction. Our groups and others have measured metabolite usage, glycolysis, mitochondrial function, and lipid metabolism in a variety of iPSC RPE model systems. While in vitro RPE models show significant promise in the discovery of disease mechanisms and therapeutic targets, there is also increasing awareness of potential limitations, including reproducibility across model systems and fidelity to native conditions. A comprehensive review of recent iPSC RPE studies shows that the most used traditional culture media are highly diverse in nutrient and metabolite content which may significantly alter RPE metabolism. Moreover, multiple types of plating substrates used could contribute to the variability in nutrient environments. A lack of consensus on baseline nutrient environments and knowledge of their impact on RPE metabolism makes comparisons between findings challenging. The goal of this proposal is to characterize the metabolic and disease-relevant phenotypic profiles of AMD iPSC RPE cells in three distinct and commonly used traditional media and physiological medium closely approximating the composition of human blood. Two AMD iPSC RPE lines and their CRISPR-corrected isogenic controls will be used in this study. RPE will be differentiated from one NIH/NYSCF AREDS2 subject iPSC line with multiple known high-risk alleles, selected to gender and complotype-match RPE lines generated from an individual with early onset macular drusen (EOMD). A splicing mutation in the CFH gene results in this severe subtype of AMD, and our preliminary data show that EOMD iPSC RPE display AMD disease-relevant features, including complement dysregulation, sub-RPE deposit formation, and altered metabolism. iPSC RPE will be cultured on twp substrates (Matrigel®, vitronectin), and maintained in four media preparations (MEM-α based, DMEM/F-12 based, X-VIVO 10TM and PlasmaxTM). This project aims to determine the impact of culture microenvironment on AMD and EOMD iPSC RPE metabolism and disease phenotype. The outcome of this project will be a new and more comprehensive understanding of how traditional and physiologic media influence the metabolic profile and phenotypic characteristics of normal and diseased RPE cells. This new understanding will aid in the interpretation of metabolite studies across model systems and help to inform the design of more physiologic cell culture media for future studies.