Defined hydrogel microenvironments for optimized neuronal culture

Three-dimensional (3D) in vitro culture systems that provide controlled, biomimetic microenvironments would be a significant technological advance for both basic cell biology research and the development of clinical therapeutics (e.g., as in vivo cell delivery constructs). A variety of signals determine cell phenotype, including those from soluble factors, immobilized biomolecules, mechanical substrates, and culture geometry. My research seeks to create hydrogel culture systems that incorporate these signals in a defined, controllable manner. Specifically, I have focused on developing hydrogels based on the extracellular matrix (ECM) component hyaluronic acid (HA) with precisely specified mechanical, chemical and geometrical microenvironments. For example, the mechanical environment presented by HA hydrogels was tuned to span the threefold range measured for neonatal brain and adult spinal cord by modifying HA with varying numbers of photocrosslinkable methacrylate groups. These hydrogels were used to evaluate the effects of mechanical properties of a 3D culture paradigm on the differentiation of ventral midbrain-derived neural progenitor cells (NPCs) and results demonstrated that the mechanical properties of these scaffolds can assert a defining influence on differentiation. In addition, whole fibronectin was incorporated into HA hydrogels as an adhesive factor to encourage angiogenesis in 3D cultures, as interplay between endothelial cells and neurons is an important determining factor during NPC development and axonal regeneration after injury. To create spatially defined neuronal cultures in three-dimensions, multiphoton excitation (MPE) was used to photocrosslink protein microstructures within HA hydrogels. This method can be used to create complex, 3D architectures that provide both chemical and topographical cues to direct cell adhesion and guidance on size scales relevant to in vivo environments. Using this approach, both dorsal root ganglion cells (DRGs) and hippocampal NPCs could be guided along user-defined, 3D paths. In future studies, these strategies can be combined into a single hydrogel to create a culture microenvironment with multiple types of highly specified cues (i.e., chemical, topographical, and mechanical).