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All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines.
1. Bioreactor preparation
- Make bioreactor molds
- Download a bioreactor CAD (Computer-aided design) file from the Supplementary CAD File or create a custom design.
- Generate a CNC (Computer Numerical Control) toolpath from the 3D model using CAM (Computer-aided manufacturing) software.
- Machine acetal molds using a CNC milling machine.
- Fabrication of bioreactors
- Mix a 10:1 base to curing agent mixture of polydimethylsiloxane (PDMS, 77 g of mixture per 4 platforms/molds).
- Place the mixture into a vacuum chamber, close all the valves, turn on the vacuum, and de-gas the mixture for at least 30 min until no air bubbles remain. Pour the mixture into molds and de-gas the molds in the vacuum chamber for 1 h.
- Close the molds with the top half of the mold in the correct orientation. Place a steel hexagonal rod over the center and clamp on both sides.
- Refill the top with PDMS.
- Cure the molds in a 65 °C oven for at least 4 h.
- Remove the platforms from the molds after cooling down to room temperature.
- Clean the devices in an ultrasonic bath using 1 h cycles of dish soap, 400 mL of 100% isopropanol, and distilled water.
- Dry overnight at 65 °C.
- Soak glass coverslips in 1% nonionic surfactant polyol for 30 min, ensuring that they do not stack so that they are all properly coated.
- Rinse well with distilled water and dry overnight at 65 °C.
- Treat the glass coverslips and PDMS platform with a plasma cleaner on high with 6 L/min of oxygen for 1-2 min. Once treated, bond the two together by pressing the PDMS down onto the coverslip for at least 30 s.
- Autoclave the bonded devices before adding cells.
2. Building an optical stimulation setup
- Using a 30 mm cage cube system, attach a 573 nm dichroic mirror in the center. Couple a red 627 nm LED (Light Emitting Diode) with a 594 nm long-pass excitation filter and attach it to the top side of the cube, with the LED facing the mirror. Then attach a blue 488 nm LED with a 546 nm short-pass excitation filter to the adjacent side of the cube, with the LED facing the mirror as shown in the schematic in Figure 1A.
- Attach a ring-actuated iris diaphragm to the bottom of the cage cube to control the size of the illuminated area.
- Power each LED with a T-Cube LED driver and control via an Arduino Uno Rev3 board. Connect the side input of the LED driver to a power source plug, connect the middle output to the Arduino, and connect the side output directly to the LEDs.
- Connect the Arduino Uno to a computer with a USB cable.
- Download files from GitHub link (https://github.com/ofvila/NMJ-function-analysis).
- Open the "Optical_Stimulation_Ramp_Arduino.ino" file from the GitHub folder.
- Set starting parameters: Steps = 30; startFrequency = 0.5; endFrequency = 3; pulseLength = 100.
- Ensure that pin output 4 is assigned to the Blue LED driver and pin output 2 is assigned to the Red LED driver. Check the middle wires of the LED driver are connected to the ground and to the respective pin output.
- Compile and load the "Optical_Stimulation_Ramp_Arduino.ino" program to Arduino.
- Connect each LED driver to its corresponding channel.
3. Cell culture setup (day -21-0)
- Primary skeletal muscle cells
- Thaw and expand primary skeletal myoblasts (obtained from Cook Myosite) for a maximum of six passages using Myotonic growth medium (+ supplement). Maintain cells in an incubator set to 37 °C and 5% CO2.
- Change the media every 2 days.
- Once cells are about 60% confluent, pass them using 0.05% Trypsin-EDTA (1x, for 5 min at 37 °C). Collect the dissociated cells and add fresh media to neutralize the trypsin.
- Spin down the cells at 300 x g and aspirate the supernatant above the cell pellet.
- Resuspend the cells with MGM and seed 1:3 with 60 mL per triple-layer flask.
- ChR2-hiPSC
NOTE: All cell lines for this work were created and used in compliance with the institutional guidelines of Columbia University, NY, USA. In this protocol, ChR2-expressing hiPSCs were generated via CRISPR-Cas9 genome editing using previously described methods18, but any stable optogenetic cell lines (lentiviral, piggyBac, etc.) can be used in the same way. Constitutive promoters expressed in both iPSCs and motoneurons were chosen (CAG). The cells were found to have healthy karyotypes, as noted in previous publications.
- Coat 6-well plates with 1 mL/well of solubilized basement membrane matrix diluted in DMEM/F12 (1:80) and incubate the plates at room temperature for 1 h.
- Seed iPSCs on coated 6-well plates with 2 mL of feeder-free cell culture medium (iPSC media), exchanging 2 mL of media every other day and passaging every 5-7 days. Maintain cells in an incubator set to 37°C and 5% CO2.
- Passaging
- Dissociate the stem cells by incubating them with 1 mL of enzyme-free stem cell-releasing reagent for 4 min and mechanically shearing them with a wide-tip P1000 pipette.
- Seed cells at a ratio of 1:24 or 1:48 in iPSC media with 2 μM of Y-27632 dihydrochloride in 2 mL/well onto coated 6-well plates.
4. Skeletal muscle tissue seeding (day -3)
- Isolate and count 30 x 106 myoblasts using an automated cell counter.
- Resuspend the myoblasts in a 4:1 mix of 3 mg/mL collagen I and solubilized basement membrane matrix (800 μL of collagen mixture + 200 μL of basement membrane matrix to reach a final volume of 1 mL). For the collagen component, add the accompanying neutralizing agent (9:1 mix of collagen to neutralizing agent) and dilute the mixture with 1x PBS to achieve a volume of 800 μL.
- Add 15 μL of cell-collagen suspension to each muscle chamber of the bioreactor, being sure to spread the suspension across both pillars using the pipette tip (Figure 2).
- Let the cell-gel mixture polymerize at 37 °C for 30 min and then fill each bioreactor well with 450 μL of Myotonic growth media.
- After 3 days (once the gel has compacted), begin myotube differentiation.
5. Myotube differentiation (days 0-14)
- Begin myotube infusion by switching tissues to 450 μL of fusion-inducing media (FS, Table 1) for 7 days, changing the media every other day.
NOTE: Try to begin myotube differentiation on the same day as motoneuron differentiation from hiPSCs in order to seed motoneurons at the end of both cell types' differentiation schedules.
- On day 7 change the media to 450 μL of maturation media Ia (MMIa, Table 1).
- On day 9, change to 450 μL of maturation media Ib (MMIb, Table 1).
- On day 11, change the media to 450 μL of NbActiv4. Keep changing the media every 2 days until the motoneurons are seeded (Step 7).
6. Motoneuron differentiation (days 0-14)
- On day 0, transfer 4 x 106 ChR2- hiPSCs to an ultra-low attachment Petri dish with 15 mL of motoneuron suspension culture medium (MSCM, Table 1). Supplement MSCM with 3 μM CHIR99021, 0.2 μM LDN193189, 40 μM SB431542 hydrate and 5 μM Y-27632 dihydrochloride.
- On day 2, isolate the neurospheres (NS) with a 37 μM reversible strainer and replate in 15 mL of MSCM with 3 μM CHIR99021, 0.2 μM LDN193189, 40 μM SB431542 hydrate, and 0.1 μM retinoic acid. After day 2, the NS should be visible in the Petri dish without using a microscope.
- On day 4, transfer the cells and media to a 50 mL tube and allow the neurospheres to settle to the bottom (5 min). Aspirate the supernatant and resuspend the cells in 15 mL of MSCM supplemented with 0.5 μM SAG, 0.2 μM LDN193189, 40 μM SB431541, and 0.1 μM retinoic acid.
- On day 7, repeat Step 6.3. but resuspend the cells in 15 mL of MSCM supplemented with 0.5 μM SAG and 0.1μM retinoic acid.
- On day 9, repeat Step 6.3. but resuspend cells in 15 mL of MSCM supplemented with 10 μM DAPT.
- On day 11, repeat Step 6.3. but resuspend the cells in 15 mL of MSCM supplemented with 20 ng/mL BDNF and 10 ng/mL GDNF.
- On day 14, seed the motoneurons into the platform.
7. Seeding motoneurons aggregates in the bioreactor (day 14)
- Prepare a 4:1 gel mixture of 2 mg/mL collagen I and Matrigel (800 μL of collagen mixture + 200 μL of Matrigel to reach a final volume of 1 mL). For the collagen component, add the accompanying neutralizing agent (9:1 mix of collagen to neutralizing agent) and dilute the mixture with 1x PBS to achieve a final volume of 800 μL.
- Use a 400 nm cell strainer to select large neurospheres and resuspend them in the gel mixture.
- Aspirate media from the reservoir and carefully from the neurosphere well (Figure 2).
- Add 15 μL of gel mixture into the neurosphere channel.
- Load a 10 μL pipette with 10 μL of gel and then pick one neurosphere.
- Deposit the NS into the neurosphere channel and ensure that the NS is in the chamber. Slowly raise the pipette while releasing the remaining gel once the NS is deposited. If unsure that the NS was correctly deposited, check its location using a microscope.
- Allow the gel to polymerize for 30 min at 37 °C.
- To the reservoirs, add 450 μL of NbActiv4 supplemented with 20 ng/mL BDNF and 10 ng/mL GDNF.
- Change the media every other day to allow for axonal growth from NS to muscle tissue.
8. Simultaneous optical stimulation and video recording of NMJ function (day 24+)
- For imaging, use an inverted fluorescent microscope with a scientific complementary metal-oxide-semiconductor (sCMOS) camera.
- Set the camera software binning to 2x2, exposure to 20 ms, rolling shutter ON, readout rate to 540 MHz, dynamic range: 12-bit & gain 1, and sensor mode: overlap.
- Use 2x objective on the microscope to image the microtissues.
- Attach a live-cell chamber (37 °C, 5% CO2) to the microscope stage.
- Select the region of interest (ROI) that contains the innervated skeletal tissues tissue to minimize the file size and processing time.
- Place a 594 nm long-pass emission filter between the sample and the imaging objective to filter out blue light pulses from the camera.
- Place a rectangular 4-well plate containing 4 bioreactors (24 tissues) into the live-cell chamber.
- Click Live View. Center and focus the image with the desired ROI.
- Upload the custom macro code from the GitHub folder (https://github.com/ofvila/NMJ-function-analysis) to control the stage position, the Arduino board, and video acquisition.
- Set output movie as day_tissue group_tissue name_experiment.nd2.
- Run the macro code with the desired X,Y coordinates set on the stage and acquire a fast time lapse with 1700 frames at 50 frames/s.
- Replace the media after imaging and return the samples to the incubator. Allow at least 24 h between image acquisition sessions to avoid tissue fatigue.
