Electrical Stimulation-Induced Differentiation of Neural Stem and Progenitor Cells
Transcript
To begin, draw patterns for individual PMMA layers and double-sided tape using appropriate software. Switch on the carbon dioxide laser scriber and connect it to a personal computer. Open the design pattern file using the software.
Place the PMMA sheets or double-sided tape on the platform of the laser scriber. Then, focus the laser onto the surface of the PMMA sheets or the double-sided tape using the Autofocus tool. Select the laser scriber as the printer. Then, use the laser scriber to start the direct ablation on the PMMA sheet or double-sided tape.
Remove the protective film from the PMMA sheets and clean the surface using nitrogen gas. For bonding together multiple layers of PMMA sheets, stack three pieces of 1-millimeter PMMA sheets and bond them under a pressure of 5 kilograms per square centimeter in a thermal bonder for 30 minutes at 110 degrees Celsius to form the flow or electrical stimulation channel assembly. Adhere 12 pieces of adapters to the individual openings in layer one of the MOE chip assembly with fast-acting cyanoacrylate glue.
Disinfect the 1-millimeter PMMA substrates, the double-sided tape, and the 3-millimeter optical-grade PMMA using ultraviolet irradiation for 30 minutes before assembling the chip. Adhere the 1-millimeter PMMA substrates on the 3-millimeter optical-grade PMMA with the double-sided tape to complete the PMMA assembly. Adhere the cleaned cover glass to the PMMA assembly with the double-sided tape.
Seal the openings of the agar bridge adapters with white finger-tight plugs. Connect the flat bottom connector to the MOE chip assembly via the medium inlet and outlet adapters. Then, connect the cone-Luer adapter to the three-way stopcocks. Add 2 milliliters of 0.01% PLL solution using a 3-milliliter syringe that connects to the three-way stopcock of the medium inlet and connect an empty 3-milliliter syringe to the three-way stopcock of the medium outlet.
Fill the cell culture regions with the PLL solution and slowly pump the coating solution back and forth. Close the two three-way stopcocks to seal the solution inside the culture regions. Incubate the MOE chip at 37 degrees Celsius overnight in an incubator filled with 5% carbon dioxide atmosphere. Open the two three-way stopcocks and flush away bubbles in the channels by manually pumping the coating solution back and forth in the channel using the two syringes.
Draw 3 milliliters of complete medium into a 3-milliliter syringe that connects to the three-way stopcock of the medium inlet, and add it to replace the coating solution in the cell culture regions. Replace the white finger-tight plug with the Luer adapter and inject 3% hot agarose to fill the adapter. Connect the Luer-lock syringe to the Luer adapter and inject 3% hot agarose through the black rubber bung to fill the Luer-lock syringe. Allow 10 to 20 minutes for the agarose to cool down and solidify.
Install the cell-seeded MOE chip onto the transparent ITO heater that is fastened on a programmable XYZ-motorized stage maintained at 37 degrees Celsius. Infuse the mNPCs by manually pumping into the MOE chip via the medium outlet. Then, incubate the cell-seeded MOE chip on the ITO heater for 4 hours.
Use electrical wires to connect an EF multiplexer to the MOE chip via the electrodes on the chip. Connect an EF multiplexer and a function generator to an amplifier to output square-wave DC pulses.
