Abstract: Calcium phosphate (CaP) deposition is characteristic of several life-threatening kidney and cardio- vascular diseases; thus, understanding CaP biomineralization is essential for developing new clinical therapies. Previous studies either analyzed extracted stones from patients or reproduced CaP deposits in quasi-2 dimensional (D) in vitro systems. However, such approaches fail to capture real-time kinetic information of mineralization and the 3D cellular microenvironments found in vivo – both of which are essential for understanding CaP biomineralization. Despite extensive research, mechanisms by which crystals nucleate, grow, and aggregate into stones are still poorly understood due to a lack of suitable bioanalytical tools that offer dynamic control over the microenvironments. Using our novel 3D microfluidic (MF)-based workbench that we achieved previously with unattained precision in controlling dynamic, spatiotemporal biological conditions of in vivo tubular systems, we will elucidate the origin and mechanisms of calcium mineralization and CaP stone formation. To recreate in vivo-like conditions, we will use an improved functional 3D-renal tubular system (RTS) using porous membranes to examine apical↔basal transport. This transformative strategy will allow us to evaluate/validate the contribution of dynamic microenvironmental cues (e.g., cellular regulations, microscale hydrodynamics) in CaP stone formation. Our overall hypothesis is that the combined interplay between tubular epithelial cells and its luminal fluid within these vivo-like RT structures would recapitulate normal and pathophysiological biomineralization by identifying precise microenvironmental dynamic factors influencing CaP stone formation. We will systematically evaluate the role of molecular factors (e.g., ionic supersaturation, stone forming activators/inhibitors) and dynamic microenvironmental cues that underlie CaP stone formation. We propose to (i) reprogram/recreate a fluidic microenvironment in improved 3D MF devices using polarized tubular epithelia, and (ii) mimic the in vivo stone-forming conditions by regulating cellular microenvironment (oxidative stress, inflammation, and fibrosis) within these 3D in vitro microsystems. Our rationale is that the sequential introduction of factor(s) of stone formation within the perfused constructs will allow us to identify currently unknown molecular bases of pathophysiological biomineralization. In Aim 1, we will validate the functional 3D-MFs by examining the endogenous (biomolecules), exogenous (environmental) factors, and the effects of inner lumen topological defects to determine factors regulating nucleation, growth, and dissolution of CaP crystals. In Aim 2, we will map and characterize real time step-by-step dynamic growth patterns/rates of crystal towards stone formation. In Aim 3, we will invoke the processes of fibrosis, inflammation, calcification, and apoptosis at controlled times to determine the impact on ...