Project Summary Access to mature cells and tissues is key to understanding the pathobiology of diseases and for developing drug therapies. Cellular and molecular studies of nerve cells are a cornerstone of modern neuroscience, yet magnocellular neurons (MCN) and some stem cell types defy conventional culture methods. For over 20 years, the challenge of culturing MCNs in vitro has been an obstacle for cellular and molecular studies on the production and regulation of oxytocin and vasopressin hormones, neurotransmitters, and their receptors. For unknown reasons, MCN cannot be grown in vitro without feeder layers. Conventional substrates have not yet been able to replace these co-dependent requirements. The goal of this proposal is to harness physical contacts and chemical signatures of the extracellular matrix to create a cell culture platform that reliably promotes, maintains, and matures MCN in vitro. We describe a two-stage approach that maximizes the progress and manages the risks while advancing innovation to solve the MCN culture challenge. First, we will test and compare brain decellularized extracellular matrix (Brain-DECM) to conventional methods to improve MCN yields beyond the established co-culture model. This will substantially reduce the time, variability, and complexity of experiments by refining the cell culture process to replace feeder-layers and decrease animal use. Because Brain-DECM is complex reagent with many unknowns, our second stage is to create a nano-scale biomimetic glial-derived extracellular matrix. The result is a tissue chip that retains the growth-permissive physical cues while incorporating developmentally-instructive biochemical ligands derived from glia. This tissue chip platform may improve neuron yield and increase access to mature MCNs to enable high-resolution imaging and analyses for difficult to culture cellular populations. We expect that this innovative solution to a long- standing challenge will be reliable, productive, and impactful to yield new opportunities for drug discovery for translational science and medicine.