More information on RPL project 2: The goal of this project in the Tulane COBRE for Sex-based Precision Medicine is to develop microphysiological systems (MPS) for modeling sex-specific pathophysiology. MPS models of human disease created with a sex-based approach will increase the relevance of mechanistic investigations and enhance the predictive accuracy of therapeutic screenings. There are currently no reports that systematically compare sex-matched and cross-sex hormone stimulation in cultured human cells. Sex-matched culture systems match the chromosomal composition of cells with known physiological hormone environments (e.g., XX cells in an estrogen-dominant environment). Cross-sex culture systems are those in which the hormone environment matches the opposite sex (e.g., XY cells in an estrogendominant environment). Focusing on cell-based models of the outer retinal microvasculature, we will first combine 2D and 3D culture assays to define the sex-specific effects of estrogen and androgen on bioenergetic capacity, vasculogenic capacity, and transcriptomic profiles determined by single cell RNA sequencing. These analyses will determine optimal hormone concentrations for quantifying the response to proinflammatory or proangiogenic stimuli in sex-matched and cross-sex MPS models of endothelial barrier function and sprouting angiogenesis. Inflammatory perturbation of endothelial barrier function and pathological neovascularization are core elements of microvascular pathologies such as diabetic retinopathy. We will use our validated MPS models of vascular permeability and angiogenesis in the outer retinal microvasculature as venues for exploring the broad hypothesis that there are sex-specific mechanisms in diabetic retinopathy. Sex-matched MPS models of the retinal microvasculature will provide novel insights regarding hormonal regulation of cytokine-induced vascular permeability and VEGF-driven sprouting angiogenesis. Cross-sex MPS models of the same processes will provide critical and currently non-existent insights regarding gender-affirming hormone therapy in the cardiovascular system. Transcriptomic signatures of sex-matched and cross-sex hormone stimulation in 2D and 3D cell cultures will be referenced to infer potential mechanisms of action in the MPS assays. We will identify candidate biomarkers to inform further research of sex- and gender-based treatments for diabetic retinopathy. Collectively, this research will deliver adaptable sex-specific human cell culture systems and methods.