Microfluidic Platforms for Focal Stimulation of Muscle Cells and Large-scale Studies of Synaptogenesis

Abstract

A central question in neurobiology is how the neuromuscular junction (NMJ) forms and maintains its high density of acetylcholine receptors (AChRs) in development. Although many of the molecules involved in NMJ synaptogenesis have been identified, the exact mechanisms by which the synaptogenic protein agrin induces and stabilizes AChR clusters remains unclear. While most in vitro experiments lack microscale control, microfluidics offers a high degree of control of the NMJ fluidic and substrate microenvironment. In this dissertation, we developed a model in which we effectively substituted the motor neuron with locally applied microfluidic agrin stimuli to subcellular regions of micropatterned myotubes. We explored two strategies for generating focal delivery: 1) a laminar flow-based hydrodynamic focusing device that operates in an open chamber, and 2) a modified Transwell that operates based on area-selective diffusion of soluble species to the basal side of myotubes. We further developed cell micropatterning methods coupled with the device designs that created well-defined arrays of muscle cultures, enabling automated microscopy and image processing protocols. Finally, we demonstrated, using the Transwell system, that focal agrin application: 1) induced localized AChR microclustering, and 2) had no effect on the stability of laminin-induced AChR pretzel clusters. Together, we present a suite of precise, user-friendly microscale tools that provide spatiotemporal control for the presentation of neuronal factors in NMJ synaptogenesis, and potentially for applications in single cell analysis and tissue engineering.