Integrated bioprinting and optogenetics platform for evaluating neural activity in stem cell-derived retinal disease models

PROJECT SUMMARY A major obstacle in studying retinal degenerative disorders lies in accurately modeling their pathophysiology from the cellular to the systems level in a benchtop lab setting. Although animal models provide valuable insight into disease processes in vivo, ethical and technical limitations often prevent the full translation of experimental findings into the clinical context. Recent advances in tissue engineering – particularly in 3D bioprinting and human induced pluripotent stem cell (hiPSC)-derived neural populations – provide new approaches for investigating neuropathology in vitro. Emerging evidence suggests that when compared to 2D substrates, biocompatible 3D hydrogel microenvironments more accurately portray normal physiologic and pathologic neural states. However, patterning 3D tissues and probing their electrical activity are not trivial and pose challenges for elucidating the relationships between cell physiology, emergent electrochemical signaling behavior, and higher- level computation and cognition. While multi-electrode arrays (MEAs), optogenetic stimulation, and voltage- sensitive fluorescent imaging are well-established techniques for interrogating native in vivo neural activity, their application towards in vitro 3D systems has been limited. This R21 project aims to develop a novel, high-throughput optical projection platform to create “visual-circuit-on- a-chip” by integrating 3D bioprinting, optogenetic stimulation, MEA recording, and real-time fluorescence imaging. In Specific Aim 1, we will develop an integrated platform for bioprinting, electrophysiology, and multiwavelength optogenetic stimulation. The bioprinting method allows for projection printing into conventional cell culture plates as well as single-well and multi-well MEA substrates. Using photopolymerizable extracellular matrix mimics, we will encapsulate hiPSC-derived neural progenitor cells and induced neurons to direct cell proliferation, neurite outgrowth, and functional connectivity. In Specific Aim 2, we will implement hiPSC technology to enable spatiotemporal control of induced multicellular differentiation and optogenetic stimulation. We will build simplified neural circuits first, then extend into a more comprehensive visual circuit platform utilizing hiPSCs engineered to produce specific populations of induced retinal neurons. Our combined technical capabilities will allow us to integrate these experimental methods into a novel all-in-one platform to yield high- throughput fabrication and interrogation of systematically patterned and stimulated biological neural networks.