This relatively simple method can help research focusing on motor neurons and neuromuscular junctions in health and disease. This protocol uses standard stem cell technology and commercially available microfluidic devices which increases reproducibility. The disconnection of motor neurons and muscle cells is an early phenomenon in several neuromuscular diseases.
A simple humanized model could help to develop strategies to counteract this phenomenon. We use this model to investigate the motor neuron disorder, amyotrophic lateral sclerosis, however, this model can also be used for other disorders in which the motor unit is essential. Begin by transferring the device using the forceps from the shipping container to the Petri dish containing 10 milliliters of 70%to 100%ethanol for sterilization for 10 seconds.
Transfer the device with forceps to a piece of paper to air dry in the Laminar Flow for about 30 minutes. Once a device is dried, use forceps to move each device to an individual 10 centimeter Petri dish. To coat the device with Poly-L-ornithine or PLO, add 100 microliters of PLO solution to the top well and observe the fluid passing from the top well through the channel to the bottom well.
Then, add 100 microliters of PLO solution to the bottom well, and repeat on the other side of the micro grooves. Finish by adding 100 microliters of PLO solution on one side to create a volume gradient between the two mirrored sides of the device to coat the micro grooves. Incubate for three hours at 37 degree Celsius and 5%carbon dioxide.
After three hours, wash the device thrice for five minutes with DPBS. Coat the device with 20 micrograms per milliliter laminin and neurobasal medium by following the procedure similar to PLO coating. Incubate the device overnight at 37 degree Celsius and 5%carbon dioxide.
The following day use a 200 microliter pipette and position the tip in the well opposite to the channel opening to remove the laminin coating from the wells. After adding DPBS to all the wells, leave the devices with DPBS in the Laminar Flow at room temperature for cell seeding. Cut the SCM sheets to the size of the device while leaving a few millimeters on each side.
After sterilizing the devices and SCM sheets in a Petri dish containing 10 milliliters of 70%to 100%ethanol for 10 seconds, transfer the devices with forceps to a six-well plate and the SCM sheets to a 10 centimeter Petri dish for air drying in the Laminar Flow. Position the device on edge to allow all sides to dry for about 30 minutes. Coat the devices by adding one milliliter of PLO solution per well to each device in the six-well plate.
Ensure the device is floating on top of the PLO solution with the channel and micro groove side facing down into the liquid. Coat the SCM sheets by adding 10 milliliters of PLO solution to the 10 centimeter Petri dish, and use the forceps to push down the SCM sheets into the liquid. Incubate devices and SCM sheets for three hours as demonstrated previously.
After three hours, wash the devices and SCM sheets twice for five minutes with DPBS followed by another wash for five minutes with sterile water. Then transfer each SCM sheet to an individual 10 centimeter Petri dish to air-dry. While working under a microscope in the Laminar Flow, use forceps to mount the silicone device with the channel and micro groove side down at a 90 degrees angle onto the SCM sheet ensuring that all sides are aligned.
Press lightly down onto the device to make sure to seal not only outer edges, but also around wells, channels, and micro grooves. To coat the device with laminin, inside the Laminar Flow and under the microscope, add 100 microliters of 20 micrograms per milliliter solution of laminin in the top well using a 200 microliter pipette. Observe the fluid passing from the top well through the channel to the bottom well without any leakage around the well and channel.
Subsequently, add 100 microliters of laminin solution to the bottom well, repeat on the other side of the micro grooves and finish with an additional 100 microliters of laminin on one side to create a volume gradient between the two mirrored sides of the device to coat the micro grooves, then incubate the device overnight. The following day, remove the coating from the wells with the 200 microliters pipette by positioning the tip in the well opposite the channel opening, after adding DPBS to all the wells, leave the devices with DPBS in the Laminar Flow at room temperature for cell seeding. To plate the neural progenitors cells or NPCs in the microfluidic device, remove DPBS from two wells on one side of the micro grooves in the device using a 200 microliter pipette.
To seed a total of 250, 000 NPCs and 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 micro grooves opposite to the freshly seeded NPCs. Add 200 microliters of day 10 motor neuron media per well to the unseeded wells.
Then add six 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 microliters pipette tip at the bottom edge of the well wall opposite the channel opening. To prevent a strong medium flow from damaging the cells on 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 micro grooves 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 chemo tactic and volumetric gradient on the 21st day of the motor neuron differentiation, add 200 microliters per well of motor neuron neurobasal 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. It is important to assess the differentiation potential of the cell lines before co-culturing them in microfluidic devices. The fusion index of MABs was determined, and about 8%were estimated to be sufficient for co-culture.
The generated motor neuron cultures were 85 to 95%positive for motor neuron markers. For characterizing and quantifying the amount of neuromuscular junctions or NMJs, the number of co-localizations between the motor neuron and pre-synaptic markers neurofilament heavy chain and synaptophysin and postsynaptic acetylene receptor marker alpha bungarotoxin, was counted through each disease stack and was normalized at a number of myosin-heavy chain labeled Myotubes present in the Z-stack. The NMJs appeared as single contact point NMJs where neurite touched upon a cluster of a subtle choline receptor at one interaction point.
Or as multiple contact point NMJs where a neurite fanned out and engaged with the a subtle choline receptor cluster over a larger surface. Quantification of the percentage of motor neuron innovated Myotubes indicated that not all Myotubes contained NMJs. Upon motor neuron activation with potassium chloride, calcium influx was observed in the Fluo-4 labeled Myotubes, which confirmed a functional connection through to motor neuron neurite in the Myotube, the addition of NMJ blocker d-tubocurarine, or DTC to the Myotube compartment resulted in an inhibition of calcium influx.
To have the largest success with this model it's incredibly important to perform a cell line quality check and to avoid the formation of air bubbles in the channels of the device. Combining the neuromuscular junction in a dish with other IPC direct cell types mimics even better the in vitro situation, this also allows to study the role of these cell types.
