Abstract: The development of tissue culture techniques by Ross Granville Harrison in 1907 has been cited as one of the ten greatest discoveries in medicine and enabled monumental advances in biological understanding. Despite the enduring importance of in vitro culture in modern biomedicine, the technology of mammalian cell culture has remained largely unchanged since the 1940's: cells are cultured on hard, flat substrates and surrounded by homogeneous solutions of medium that do little to recreate the exquisite microenvironments found in vivo. Cells are well known to respond to multiple cues found within their in vivo niches, e.g., concentration gradients of soluble and tethered biochemicals, matrix rigidity, patterns of matrix ligands, and interactions with other cell types;however, few methods exist to recapitulate these cues in in vitro cell culture studies. To address these limitations, I propose creating versatile, three-dimensional in vitro niches with precise spatial and temporal resolution of cellular cues. These three-dimensional microenvironments will be fabricated using innovative and transdisciplinary approaches that combine advances in protein engineering, biomaterials, and microfluidics with traditional cell biology protocols. As a model system, these in vitro niches will be used to quantitatively study the cellular biomechanics and signaling mechanisms regulating neural progenitor cell (NPC) migration. NPC chemotaxis within gradients of soluble factors is hypothesized to be contextual and reliant on additional biomechanical cues from the 3D matrix. The presence of NPCs within specific niches of the brain opens up the tantalizing possibility that the adult central nervous system may be able to regenerate following injury or disease if NPCs were induced to migrate to sites of need. The development of quantitative, in vitro mimics of in vivo niches will have a profound impact on biomedical research by enabling scientists to test entirely new hypotheses about the interactions between different cells and their three-dimensional microenvironments. Public Health Relevance: Despite the enduring importance of tissue culture techniques in modern biomedicine, the technology of mammalian cell culture has remained largely unchanged since the 1940's: cells are cultured on hard, flat substrates and surrounded by solutions of medium that do little to recreate the exquisite microenvironments (called niches) found inside the body. To address these limitations, I propose creating versatile mimics of three-dimensional niches with precise spatial and temporal resolution of cellular cues. As a model system, these engineered niches will be used to quantitatively study the cellular biomechanics and signaling mechanisms regulating neural progenitor cell (NPC) migration, opening the door to future therapies for regeneration of the central nervous system.