Assessing a Functional Neuromuscular Junction Via Simultaneous Optical Stimulation and Video Recording

Begin with a bioreactor with a muscle chamber containing collagen-embedded muscle tissue supported over the chamber's pillars.

The neurosphere channel contains a neurosphere in a collagen matrix. The motor neurons of the neurosphere express light-sensitive channels.

Add a differentiation medium containing neural growth factors that stimulate the neurons to extend their projections.

Regularly exchange the medium to ensure continuous neuron extension toward the muscle tissue, forming neuromuscular junctions.

Observe the bioreactor under a microscope equipped with a camera and a blue light source.

Apply blue light pulses to open the light-sensitive channels in the neurons, allowing ions from the medium to flow and stimulate the neurons to generate electrical signals.

These signals travel via the neuronal projections to the neuromuscular junctions, transmitting the electrical signal to the muscle tissue.

Record a video simultaneously. A gradual contraction of the muscle fiber confirms the development of a functional neuromuscular junction.

Prepare a 4-to-1 mix of 3 milligrams per milliliter collagen I and solubilized basement membrane matrix. Then, resuspend the myoblasts in this freshly prepared collagen mixture.

Next, add 15 microliters of this cell-collagen suspension to each muscle chamber of the bioreactor, making sure to spread the suspension across both pillars using the pipette tip. Allow the cell gel mixture to polymerize at 37 degrees Celsius for 30 minutes. Then, fill each bioreactor well with 450 microliters of myotonic growth media.

On day 11 of motor neuron differentiation, transfer the cells and media to a 50-milliliter tube, and allow the neurospheres to settle to the bottom for 5 minutes. Aspirate the supernatant and resuspend the cells in 15 milliliters of motor neuron suspension culture medium supplemented with 20 nanograms per milliliter of brain-derived neurotrophic factor and 10 nanograms per milliliter of glial-cell-derived neurotrophic factor.

On day 14 of the differentiation protocols, seed the motor neurons into the platform. Prepare a 4-to-1 gel mixture of 2 milligrams per milliliter collagen I and solubilized basement membrane matrix. Use a 400-nanometer cell strainer to select large neurospheres, and resuspend them in the prepared gel mixture.

Carefully, aspirate media from the reservoir and the neurosphere well. Then, add 15 microliters of the gel mixture into the neurosphere channel. Load a 10-microliter pipette with 10 microliters of gel, and pick one neurosphere. Deposit the neurosphere into the neurosphere channel, ensuring that it is in the chamber. Then, slowly raise the pipette while releasing the remaining gel once the neurosphere is deposited.

Allow the gel to polymerize for 30 minutes at 37 degrees Celsius. Then, add 450 microliters of NbActiv4 supplemented with 20 nanograms per milliliter of brain-derived neurotrophic factor and 10 nanograms per milliliter of glial-cell-derived neurotrophic factor to the reservoirs.

For imaging, use an inverted fluorescent microscope with a scientific complementary metal oxide semiconductor camera software. Set the binning to 2-by-2, exposure to 20 milliseconds, rolling shutter on, readout rate to 540 megahertz, dynamic range to 12-bit and gain 1, and sensor mode to overlap. Use a 2x objective on the microscope to image the microtissues, and attach a live-cell chamber to the microscope stage.

Select the region of interest that contains the innervated skeletal tissues to minimize the file size and processing time. Then, place a 594-nanometer long-pass emission filter between the sample and the imaging objective to filter out blue light pulses from the camera. Next, place a rectangular four-well plate containing four bioreactors into the live-cell cell chamber. Then, click Live View, and center, and focus the image with the desired ROI.

Download the custom macrocode from the GitHub folder to control the stage position, the Arduino board, and video acquisition. Then, set the output movie as day_tissuegroup_tissuename_experiment.ND2. Run the macrocode with the desired x- and y-coordinates set on the stage, and acquire a fast time-lapse lapse with 1,700 frames at 50 frames per second.

After imaging, replace the media and return the samples to the incubator. Allow at least 24 hours between image acquisition sessions to avoid tissue fatigue.