The initiators and/or drivers of Age-related Macular Degeneration (AMD) remain elusive. Current knowledge suggests that interventions in late disease stages may not be as effective as earlier in the disease due to a “domino effect”, wherein there are many inflammatory factors acting later in the disease. We posit that there is value in doing proof of mechanism studies to identify biological effects at the early stage of disease. The major genetic risk factors include CFH on chromosome1 and ARMS2/HTRA1 on chromosome 10. Despite genetic heterogeneity, most subjects with AMD show common early pathology that include drusen formation and complement activation. Based on these common features, the core structures affected in AMD that require further investigation are the RPE, Bruch’s membrane (BrM) and the choroid. The recent discovery of induced pluripotent stem cell (iPSC) tissue models to study disease mechanisms and the ability to assemble 3D tissues permit the study of cell-cell interactions. We have designed a 3D tissue chip which includes an RPE monolayer sitting on a choroid containing endothelial cells, fibroblasts and pericytes which eventually form a BrM to complete the structure. Since the endothelial cells undergo anastomosis the final choroidal vasculature can be perfused to more closely mimic the in vivo environment. Our current goal is to utilize this model to characterize the responses of the RPE-choroid to various environmental stressors under the influence of the CFH and ARMS2/HTRA1 risk genotypes. Please note, not only can we assemble the 3D oBRB from different donors, but we can subsequently analyze four different parts; the supernatant in the top compartment (i.e., sub-retinal space), the flowthrough in the bottom compartment (i.,e, blood supply of the choroid), as well as the two tissue compartments, RPE/BrM and CC. Aim 1 will characterize the perfused 3D RPE/choroid chip and provide baseline data for our readouts (gene expression patterns based on scRNA-Seq; extracellular and intracellular complement activation; BrM formation over time; and extracellular vesicle analysis). In Aim 2, we ask how these parameters are affected genetic risk factors, assembling the RPE/choroid complex from iPSC cells using a combination of RPE-risk with choroid-non-risk or vice versa. And finally, in Aim 3, we characterize the response of the perfused 3D RPE/choroid chip to AMD relevant stressors, smoke, fatty acid exposure and alteration in flow rate. Overall, our data will provide unique knowledge about the influence of genetic variation on complement secretion, expression and synthesis of intracellular and extracellular complement from pre- drusen to post-drusen development, and the proposal’s outcomes will fill a critical gap in our understanding of genetic variants on RPE complement activity and identify new potential therapeutic targets directed at the RPE in the early stages of AMD.