Figure 1.

Schematic of neuromuscular junction microfluidic device. A) Primary myoblasts and mouse embryonic stem cell-derived motor neurons were seeded in separate cell culture compartments in a microfluidic device via the cell seeding ports. Myoblasts were then differentiated (over two days), forming multinucleated myofibers. During this period, motor neurons were kept fluidically isolated. Motor neuron processes were then recruited across the microchannel, via a chemotactic gradient of GDNF and BDNF (days 4 and 5) generated by small but sustained fluid flow from the muscle to the motor neuron compartment. NMJs were allowed to mature until day 10, under static conditions. B) Fluorescent image of microfluidic device showing motor neurons in the left-hand side compartment and muscle fibers in the right-hand side after 6 days of culture. Muscle fibers were stained for actin (green) and motor neurons were visualized by the Hb9:GFP reporter (green). Nuclei were stained with DAPI (blue). Scale bar represents 200 μm. C) A close-up fluorescent image of neurite processes crossing the central microchannels. Scale bar represents 50 μm. D) A close-up fluorescent image showing a neurite process extending, branching and contacting a muscle fiber. Scale bar represents 50 μm.