This study demonstrates a microfluidic approach to investigate axonal transport in developing motor neurons derived from mouse embryonic spinal cord tissues. By creating a concentration gradient of growth factors, the axons are directed towards distal wells, allowing for real-time imaging of organelle movement.
Begin with a polymer-coated microfluidic chamber with proximal and distal wells connected via multiple microgrooves.
In the proximal well, place mouse embryonic spinal cord tissues rich in developing motor neurons.
Supplement this well with a nutrient medium. Incubate to allow neuron growth and adherence to the polymer-coated surface.
Later, add a growth factor-free nutrient medium to the proximal well.
In distal wells, add a higher volume of nutrient medium containing growth factors to create concentration and volume differences.
These differences cause the axons, the longer neuronal projections, to grow toward the distal wells via the microgrooves.
Add a fluorescent dye solution into both wells.
The dye molecules enter the neurons and stain the mitochondria and acidic organelles at both ends.
In live confocal imaging, fluorescently stained organelles move in both directions between the neural cell bodies and the axons, confirming axonal transport in motor neurons.
Dispose of all medium from the proximal compartment of the MFC. Pick up a single spinal cord explant with a pipette in a total volume of four microliters and inject it as close as possible to the cave. Draw out any excess liquid from the proximal well via the lateral outlets. Repeat the previous step two more times, and make sure that the explants are embedded in the proximal channel. Slowly, add 150 microliters of SCEX medium to the proximal well.
One day after plating, replace the SCEX medium in the proximal compartment, and add rich SCEX medium to the distal compartment, maintaining a volume gradient of at least 15 microliters per well between the distal and proximal wells. After four to six days, the axons should cross to the distal compartment and be ready for axonal transport imaging.
To label the mitochondria and acidic compartments, prepare fresh SCEX medium with 100-nanomolar Mitotracker deep red FM and 100 nanomolar LysoTracker red, and add it to the microfluidic chambers. Incubate them for 30 to 60 minutes at 37 degrees Celsius, then wash three times with warm SCEX medium. Proceed with live imaging of axonal transport, acquiring a 100-time lapse image series at 3-second intervals with a total of 5 minutes per movie.
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