Generating Neuromuscular Junctions Using a Microfluidic Device

Begin with a polymer-coated microfluidic device featuring interconnected wells carrying mirrored compartments that are connected via microgrooves.

Seed neural progenitor cells into the wells of one compartment.

Allow them to migrate into the interconnected channel and adhere to the polymer.

Overlay with a growth medium to promote neuronal maturation into motor neurons.

In the opposite compartment, seed the wells with muscle progenitor cells, which migrate and adhere to the interconnected channel.

Replace the medium with a differentiation medium, inducing the fusion of muscle progenitor cells and forming multinucleated muscle cells.

Add neuron-differentiation medium containing growth factors to the muscle cell-containing compartment.

Meanwhile, add a half volume of growth factor-free medium to the neural cell-containing compartment.

The growth factors and a higher volume of medium in the opposite compartment promote the motor neurons to extend their processes through the microgrooves.

These neuronal processes then connect with the muscle cells, forming neuromuscular junctions.

To plate the neural progenitor cells, or NPCs, in the microfluidic device, remove DPBS from two wells on one side of the microgrooves in the device using a 200-microliter pipette. To seed a total of 250,000 NPCs in 60 to 100 microliters of day 10 motor neuron medium per device in the top right well, seed half of the cell suspension close to the channel opening at an angle of 45 degrees.

Pause for a few seconds to allow the cell suspension to flow through the channel before adding the remaining half of the cell suspension in the lower well. Use a pen to mark the seeded side as NPC or equivalent for easy orientation of the device without a microscope and incubate for five minutes for cell attachment. Next, slowly top up the two NPC-seeded wells with an additional day 10 motor neuron medium to a total volume of 200 microliters per well. Using a 200-microliter pipette, remove DPBS from the two wells on the other side of the microgrooves opposite to the freshly seeded NPCs.

Add 200 microliters of day 10 motor neuron media per well to the unseeded wells. Then, add 6 milliliters of DPBS per 10-centimeter dish around the device to prevent evaporation of the medium during incubation.

To change the medium, slowly remove media in both wells with NPCs by positioning the 200-microliter pipette tip at the bottom edge of the well wall opposite the channel opening. To prevent a strong medium flow from damaging the cells in the channel, slowly, add 50 to 100 microliters of fresh motor neuron medium to each well, by continuously changing between the top and bottom well. Repeat the process until each well contains 200 microliters of medium.

On day 17 of the motor neuron differentiation, remove the motor neuron medium on the unseeded side of the microgrooves in the device with a 200-microliter pipette and wash the wells with DPBS. Seed a total of 200,000 human primary mesoangioblasts or MABs in 60 to 100 microliters of growth medium per device by seeding half of the cell suspension in the top well.

Pause for a few seconds to allow the cell suspension to flow through the channel before seeding the remaining half of the cell suspension in the bottom well, as demonstrated earlier for plating NPCs. Incubate the device for five minutes for cell attachment. Then, top up the two freshly MAB-seeded wells with an additional growth medium and incubate again as demonstrated.

To initiate the chemotactic and volumetric gradient on the 21st day of the motor neuron differentiation, add 200 microliters per well of motor neuron basal medium containing growth factors to the myotube compartment. Then, add 100 microliters per well of motor neuron basal medium without growth factors to the motor neuron compartment.