This study presents a protocol for the differentiation of neural precursor cells solely induced by direct current pulse stimulation in a microfluidic system. This technique provides a beneficial approach for reducing the experimental setup time, sample volume, and reagent volume. Furthermore, the MOE chip is suitable for confocal microscopy observations.
The simple direct current pulse treatment can control mouse neural precursor cells'fate and could be used to develop therapeutic strategies for nervous system disorders. To begin, draw patterns for individual PMMA layers and the double-sided tape using appropriate software. Switch on the carbon dioxide laser scriber and connect it to a personal computer.
Open the designed 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 auto-focus 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 one millimeter PMMA sheets and bond them under a pressure of five 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 one millimeter PMMA substrates, the double-sided tape, and the three millimeter optical grade PMMA using ultraviolet irradiation for 30 minutes before assembling the chip. Adhere the one millimeter PMMA substrates on the three millimeter optical grade PMMA with the double-sided tape to complete the PMMA assembly. Adhere the cleaned coverglass 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 two milliliters of 0.01%PLL solution using a three milliliter syringe that connects to the three-way stopcock of the medium inlet, and connect an empty three 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 three milliliters of complete medium into a three 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 bong 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 four 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.
Remove all the flat bottom connector and adapters on the MOE chip and fill the adapters with PBS, then seal the adapter openings with parafilm. After immunostaining, observe the cells using a confocal fluorescence microscope. The DC pulse stimulation resulted in simultaneous mNPCs differentiating into neurons, astrocytes, and oligodendrocytes in stem cell maintenance medium.
The percentages of neurons, astrocytes, and oligodendrocytes in the control group and in the stimulation group are shown here. After the DC pulse treatment, the mNPCs expressed significantly high numbers of neurons at DIV seven, astrocytes at DIV three, and oligodendrocytes at DIV seven and DIV 14 were present at relatively higher levels in the stimulation groups than in the control group. Neurospheres formed by 30 to 40 cells are preferred for initiating mNPC differentiation.
Overgrowth of mNPCs will impair cell survival during the differentiation process.
