Poster Y10 - Embryonic Development On-chip: Engineering Neuromuscular Circuitry

Introduction: Human locomotion results from precisely coordinated processes between two interdependent tissues: nerve and muscle. Intertwined morphogenic signals that vary in space and time, autonomously driven cellular responses, and electromechanical inputs play a role in the carefully orchestrated dance of neuromuscular tissue development. A consequence of this complex process is multiple means through which normal development can be disrupted, resulting in neuromuscular disease. Furthermore, these intricacies pose challenges for researchers trying to identify the causes of such diseases or to characterize the processes required for normal development. Traditional methods and tools, such as model organisms and tissue culture, have long been invaluable in expanding our understanding of each tissue’s independent developmental processes, and form the basis upon which new experimental techniques, such as the one presented here, are derived.

Here, we present an innovative, microfabricated 3D co-culture platform that blends many of the dynamic aspects of the in vivo environment with the ease and control of an in vitro model system. This device allows for the independent development of precursors into two cell types on-chip, by providing each with its requisite morphogens, while simplifying the communicative landscape between the cells to interconnecting microchannels. This unique culturing method enables us to probe neuromuscular-specific, paracrine cell-cell signaling pathways that contribute to a wider network of signals from other tissues within the developing organism. Ongoing research seeks to characterize and prove the functionality of neuromuscular network formation within the device, prior to investigations into disease modeling, nerve-muscle pairing, and motor neuron subtype specification.

Materials and

Methods: Overall microdevice geometry was based on previous work by Demers et al, 2016 and fluidic delivery was modeled using COMSOL Multiphysics simulations. Devices were fabricated using dual-sided photolithography and deep reactive ion etching of a silicon wafer, followed by anodic bonding to seal the fluidic channels and attachment of inlets/outlets. Hydrogel-encapsulated cells are then loaded into spatially separated culturing chambers, to which the fluidic systems deliver requisite media. Hb9:GFP mouse embryonic stem cells are provided neuronal media supplemented with small molecules to induce motor neuron formation on one side; C2C12 myoblasts are provided myogenic media to induce myotube formation on the other. The current aim is to generate functional neuromuscular junctions from the developing tissues on-chip and is being assessed using immunofluorescence staining and electrophysiological techniques.

Colocalization of neuron-specific beta-tubulin (neurons), myosin (muscle), and alpha-bungarotoxin (junction) immunostains is indicative of neuromuscular junction formation. To assess functionality, a stimulation-contraction assay is used in which muscle is monitored for contraction following the excitation of neurons in the adjacent chamber. For this technique to provide conclusive results, neurons and muscle must be independently tested to ensure they are capable of the expected physiological activity. Thus, FM2-10 exocytosis dye is used to measure the release of acetylcholine from mESC-derived neurons when challenged with a chemical stimulant; video-based microscopy imaging is used to detect the movement of live, fluorescently labeled myoblasts in response to acetylcholine application. Additionally, COMSOL Multiphysics simulations are employed to visualize the spatial restriction of agents applied to each independent cell population.

Results, Conclusions, and Discussions: Devices have been successfully fabricated and used for cell culture. GFP fluorescence in the motor neuron chamber indicates successful differentiation of cultures on-chip and live membrane labeling of C2C12 precursors shows progression from myoblasts to fused myotubes. mESC-derived neurons longer than one millimeter in length have been observed, indicating that the culturing conditions are sufficient for axon elongation into the adjacent muscle chamber. On-going experimentation using immunocytochemistry to label pre- and post-synaptic structures is expected to show axon outgrowth results in nerve terminal colocalization with muscle targets. If outgrowth is insufficient in length or direction, chemoattractive factors can be doped within the hydrogel matrix of the interconnecting channels to recruit axons toward the adjacent chamber.

Fluorescent and brightfield imaging confirms that C2C12s are capable of contraction both spontaneously and with the application of stimulants. Neuron exocytosis studies are currently underway and are expected to confirm that the culture conditions are sufficient for electrophysiological activity. COMSOL Multiphysics simulations show that the application of chemical stimulants in one chamber results in linear decay across the microchannels, restricting the impact of the chemical to a single cell population. Taken together, these checkpoints show that the embryonic development of functional neuromuscular circuitry is feasible within this platform. Lastly, to finalize the characterization of the device for more sophisticated studies, we expect to complete experiments showing muscle contraction in response to stimulated neuron firing.

This device offers the user significant control over the spatiochemical landscape of developing cell types from the stem cell stage to the connection and maturation of two tissue types. It is therefore uniquely suited to understanding the chemical and temporal complexity of the embryological landscape. By probing the culture at various time points, introducing various morphogen or drug gradients, or loading the chambers with cells exhibiting disease phenotypes, this platform can be used to probe many exciting new avenues.

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