Project Summary In addition to significant human distress, neurological disorders cost the U.S. economy more than $1.5 trillion per year—8.8 percent of the gross domestic product. This physical, emotional and financial burden underscores the potential benefit from developing innovative platforms to study brain development, physiology, and associated diorders. The advent of human induced pluripotent stem cell (hiPSC)-derived 3D cortical organoid cultures has shown great promise as a model system, yet there remain a number of technical limitations that have stymied their ability to recapitulate critical non-cell autonomous aspects of human brain development. These components include extrinsic influences of the extracellular matrix (ECM), skull, and axial morphogen gradients that together help shape and diversify regions of the developing brain. Currently, standard organoid protocols involve embedding organoids in Matrigel droplets or in suspended bath culture, which prevents reproducible user-control of the extracellular environment. This limits the ability to create morphogen gradients that topographically polarize stem cells, or to ask how molecular and physical properties of the ECM outside the brain parenchyma guide neurodevelopment. To address these challenges, we propose developing 3D bioprinted cortical organoid constructs that recapitulate key microenvironmental cues of native brain tissue. This project builds upon our recent technological achievements, enabling embedded bioprinting of custom tissue constructs at high spatial resolution (20 µm) specifically designed for long-term organoid culture. Our goal is to bioprint cortical brain organoids into 3D scaffolds with customizable molecular and physiomechanical compositions. The synergy of brain organoid and bioprinting technologies provides a nearly unlimited potential to manipulate extrinsic developmental cues of a complex multicellular human model system. We will pursue two integrated Specific Aims using the multi-PI leadership mechanism to combine complementary research skills and expertise in the Sloan (neurodevelopment) and Serpooshan (tissue engineering) laboratories. In Aim 1, we will decouple the molecular composition and physical stiffness paramaters of the ECM, and ask how these factors influence neural development, differentiation, maturation and architecture. In Aim 2, we will use three separate approaches to generate a stable morphogen gradient within bioprinted constructs, which we will use to induce intra-organoid polarization of both dorsal (pallial) and ventral (subpallial) regional identities. Together, these approaches offer a novel platform for 3D stem cell modeling that could be applied broadly to numerous systems and usher a new generation of neurodevelopmental modeling.