Microtissue Engineered Neural Networks As Optically-Controlled Living Electrodes For Circuit Modeling And Neuroprosthetics

Dayo Adewole, University of Pennsylvania

Abstract

Neural interfaces transmit signals between the nervous system and an external device throughthe activation of neuronal circuits (stimulation) or detection of neuronal activity (recording). Beyond research, these devices are used medically to restore or approximate neurological function, often following injury or disease—e.g., cochlear implants to provide auditory perception. However, current implantable neural interfaces use inorganic, rigid electrodes, inducing a foreignbody response that limits their practical use for chronic medical applications. In this dissertation, we applied tissue engineering and optogenetic techniques to develop the first living, implantable microtissue engineered neural networks (“μTENNs”) that can be controlled and monitored with light as “living electrodes”. These microtissues were fabricated with discrete, spheroidal cell populations (“aggregates”) of multiple neuronal subtypes that projected long axons through an extracellular matrix (ECM) within a protective hydrogel cylinder (“microcolumn”). Fabrication and culture methodology were developed to optimize μTENN architecture and health. Longitudinal imaging, analysis, and immunocytochemistry of μTENNs were conducted to evaluate growth, viability, and network structure in vitro across multiple parameters including microtissue length and polarity. These studies also demonstrated that the aggregate fabrication method reproducibly generated μTENNs closely mimicking the connectome—i.e., locally connected circuits spanned by long axonal tracts. Calcium imaging and network analysis of single μTENNs and assemblies of multi-μTENN “chains” over time characterized their functional development and emergent network-level properties across neuronal subtypes, with photostimulation demonstrating that μTENN activity could be driven using optical input. Further, multi-μTENN studies established proof-of-concept for a modular approach to create and probe complex in vitro models of neural circuits. Finally, μTENNs expressing optical reporters were transplanted in rodent primary visual and auditory cortex and monitored through a transparent cranial window as early validation of the living electrode strategy in vivo. Immunohistochemistry demonstrated that living electrodes survived, projected neurites into host tissue, and maintained sufficient viability and structure to provide optical output following transplant. In conclusion, this dissertation developed and characterized implantable living electrodes that may be built with a variety of cell types and controlled/monitored with light. These studies lay the foundation for optobiological modeling of the connectome and neural interfacing with biologically-based devices.