Emergence of functional neuromuscular junctions in an engineered, multicellular spinal cord-muscle bioactuator

A novel, engineered platform for the co-culture of 3D muscle strips innervated by an intact spinal cord central pattern generator (CPG) is a significant advancement over traditional 2D cell culture, allocellular biological robots, and as a translational platform for pharmacological testing. Development of this platform requires an interdisciplinary approach bridging fields as diverse as tissue engineering, nanotechnology, materials science, and soft robotics. We confirmed the presence of robust neuronal and glial growth from a cultured segment of intact lumbar spinal cord. For the first time, we also demonstrated the sustained electrical activity of a cultured spinal cord out to approximately 2 weeks in vitro. At 0 DIV, the spinal cord was highly active at baseline and the addition of glutamate reduced the overall firing rate at both 10 µM and 200 µM doses. By ~7 DIV, the spontaneous firing at baseline was greatly reduced, but the spinal cord still became highly active in response to both low and high doses of glutamate. At ~14 DIV, spinal cords did not exhibit spontaneous firing at baseline or respond to a low dose of glutamate. However, a high dose of glutamate initiated robust electrical responses with a clear bursting pattern indicative of a pattern generating circuit. A spinal cord was co-cultured in an organotypic manner with an engineered 3D C2C12-derived skeletal muscle biobot where spinal motor neurons co-localized with post-synaptic acetylcholine receptors (AChR) clusters to form motor endplates at a time-scale developmentally similar to in vivo. The spinal cord is capable of inducing spontaneous contraction of the muscle via these NMJs. Glutamate stimulation of the spinal cord elicited patterned contractions from spinal central pattern generators (CPGs) which were blocked by application of glutamate receptor antagonists. Primary skeletal muscle was determined to have larger myotube diameters, AChR clusters had greater surface area, and demonstrated more consistent spontaneous contractions than their C2C12 counterparts. A new platform built for multiple 3D muscle strips was developed and optimized. These muscle cells exhibited spontaneous contractions and were capable of matching a variety of electric stimulus frequencies with differing forces of contraction. Immunoblotting revealed that though the extracted primary tissue was a mix of Type I and Type II fibers, this identity was not retained by the dissociated satellite cells when cultured on Matrigel-coated glass in 2D. When grown in mixed skeletal muscle-spinal cord conditioned media, primary skeletal muscle cultures regained a mixed Type I/Type II fiber-type identity, which emphasizes the important role of diffusible factors released from the spinal cord in muscle development. This platform allows for the study of basic research questions about the development of each component of the neuromuscular junction through high-resolution fluorescent imaging of disease mutant models in an in vitro environment closer to the physiological reality than ever before. Better understanding of the neuromuscular junction and its components will inform future designs of biobots, prosthetics, pharmaceuticals, and potentially even lab-grown meats.U of I OnlyAuthor requested U of Illinois access only (OA after 2yrs) in Vireo ETD syste