electrical stimulation-induced differentiation of neural stem and progenitor cells

Electrical Stimulation-Induced Differentiation of Neural Stem and Progenitor Cells

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.

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1. Design and fabrication of the multichannel, optically transparent, electrotactic (MOE) chip
<ol>
	<li>Draw patterns for individual polymethyl methacrylate (PMMA) layers and the double-sided tape using appropriate software (Figure 1A, Table of Materials). Cut both the PMMA sheets and the double-sided tape with a CO2&#160;laser machine scriber (Figure 1B).
	<ol>
		<li>Switch on the CO2&#160;laser scriber and connect it to a personal computer. Open the designed pattern file using the software.</li>
		<li>Place the PMMA sheets (275 mm x 400 mm) or double-sided tape (210 mm x 297 mm) on the platform of the laser scriber (Figure 2A). Focus the laser onto the surface of the PMMA sheets or the double-sided tape using the auto-focus tool.</li>
		<li>Select the laser scriber as the printer, and then &#34;print&#34; the pattern using the laser scriber to start the direct ablation on the PMMA sheet or double-sided tape and obtain individual patterns on the PMMA sheet or tape (Figure 2B).</li>
	</ol>
	</li>
	<li>Remove the protective film from the PMMA sheets and clean the surface using nitrogen gas.&#160;&#160;&#160;&#160; 
	NOTE: The drawing of the PMMA pattern and direct machining of the PMMA sheet were performed according to a previous report.</li>
	<li>For bonding together multiple layers of PMMA sheets, stack three pieces of 1 mm PMMA sheets (Layers 1, 2, and 3), and bond them under a pressure of 5 kg/cm2&#160;in a thermal bonder for 30 min at 110 &#176;C to form the flow/electrical stimulation channel assembly (Figure 2C).&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; 
	NOTE: Different batches of commercially obtained PMMA sheets have slightly different glass transition temperatures (Tg). The optimal bonding temperature needs to be tested at 5 &#176;C increments close to the Tg.</li>
	<li>Adhere 12 pieces of adaptors to the individual openings in Layer 1 of the MOE chip assembly with fast-acting cyanoacrylate glue.&#160;&#160;&#160;&#160;&#160;&#160; 
	NOTE: The adaptors are made of PMMA by injection molding. The flat surfaces at the bottom are for connecting to the MOE chip. The adaptors bearing 1/4W-28 female screw threads are for connecting white finger-tight plugs, flat bottom connectors, or Luer adaptors. Be careful when using fast-acting cyanoacrylate glue. Avoid splashing into the eyes.</li>
	<li>Disinfect the 1 mm PMMA substrates (Layers 1-3), the double-sided tape (Layer 4), and the 3 mm optical grade PMMA (Layer 5) using ultraviolet (UV) irradiation for 30 min before assembling the chip (Figure 1A).</li>
	<li>Adhere the 1 mm PMMA substrates (Layers 1-3) on the 3 mm optical grade PMMA (Layer 5) with the double-sided tape (Layer 4) to complete the PMMA assembly (Layers 1-5) (Figure 1A).</li>
	<li>Prepare the clean cover glass for the assembly on the chip.
	<ol>
		<li>Fill a ten-fold dilution of the detergent in a staining jar (see the&#160;Table of Materials), and clean the cover glass in this detergent using an ultrasonic cleaner for 15 min.</li>
		<li>Thoroughly rinse the staining jar under running tap water to remove all traces of the detergent.</li>
		<li>Continue rinsing with distilled water to remove all traces of tap water and repeat step 1.7.2 two times.</li>
		<li>Dry the cleaned cover glass by blowing it with nitrogen gas.</li>
	</ol>
	</li>
	<li>Disinfect the PMMA assembly (Layers 1-5), the double-sided tape (Layer 6), and the cover glass (Layer 7) using UV irradiation inside a biosafety cabinet for 30 min before assembling the chip (Figure 1A).</li>
	<li>Adhere the cleaned cover glass (Layer 7) to the PMMA assembly (Layers 1-5) with the double-sided tape (Layer 6) (Figure 1A).</li>
	<li>Incubate the MOE chip in a vacuum chamber overnight; use the MOE chip assembly for subsequent procedures (Figure 3).</li>
</ol>
2. Coating poly-L-lysine (PLL) on the substrate in the cell culture regions
<ol>
	<li>Prepare the polytetrafluoroethylene tube, flat-bottom connector, cone connector, cone-Luer adaptor, white finger-tight plug (also called stopper), Luer adaptor, Luer lock syringe, and black rubber bung (Figure 4A, Table of Materials). Sterilize all the above components in an autoclave at 121 &#176;C for 30 min.</li>
	<li>Seal the openings of the agar bridge adaptors (Figure 1A) with the white finger-tight plugs. Connect the flat-bottom connector to the MOE chip assembly via the medium inlet and outlet adaptors (Figure 4B). Connect the cone-Luer adaptor to the 3-way stopcocks.</li>
	<li>Add 2 mL of 0.01% PLL solution using a 3 mL syringe that connects to the 3-way stopcock of the medium inlet (Figure 4B-<img alt="Equation 1" src="/files/ftp_upload/22350/22350eq01.jpg" style="margin: auto;" />).</li>
	<li>Connect an empty 3 mL syringe to the 3-way stopcock of the medium outlet (Figure 4B-<img alt="Equation 2" src="/files/ftp_upload/22350/22350eq02.jpg" style="margin: auto;" />).</li>
	<li>Fill the cell culture regions with the PLL solution. Manually pump the coating solution back and forth slowly. Close the two 3-way stopcocks to seal the solution inside the culture regions.</li>
	<li>Incubate the MOE chip at 37 &#176;C overnight in an incubator filled with 5% CO2&#160;atmosphere.</li>
</ol>
3. Preparation of the salt bridge network
<ol>
	<li>Following step 2.6, open the two 3-way stopcocks and flush away the bubbles in the channels by manually pumping the coating solution back and forth in the channel using the two syringes.</li>
	<li>Draw 3 mL of complete medium (stem cell maintenance medium consisting of Dulbecco&#39;s modified Eagle&#39;s medium/Ham&#39;s nutrient mixture F-12 (DMEM/F12), 2% B-27 supplement, 20 ng/mL EGF, and 20 ng/mL bFGF) into a 3 mL syringe that connects to the 3-way stopcock of the medium inlet (Figure 4B-<img alt="Equation 1" src="/files/ftp_upload/22350/22350eq01.jpg" style="margin: auto;" />&#160;and&#160;Figure 4B-<img alt="Equation 3" src="/files/ftp_upload/22350/22350eq03.jpg" style="margin: auto;" />).</li>
	<li>Add 3 mL of complete medium to replace the coating solution in the cell culture regions. Connect an empty 5 mL syringe to the 3-way stopcock of the medium outlet (Figure 4B-<img alt="Equation 4" src="/files/ftp_upload/22350/22350eq04.jpg" style="margin: auto;" />).</li>
	<li>Prepare the salt bridge network (Figure 5).
	<ol>
		<li>Cut the black rubber bung to produce a gap and insert the silver (Ag)/silver chloride (AgCl) electrodes through the black rubber bung and into the Luer lock syringe (Figure 4A).</li>
		<li>Replace the white fingertight plug with the Luer adaptor, and inject 3% hot agarose to fill the Luer adaptor. 
		NOTE: For the preparation of the hot agarose, dissolve 3 g of agarose powder in 100 mL of phosphate-buffered saline (PBS) and sterilize in an autoclave at 121 &#176;C for 30 min.</li>
		<li>Connect the Luer lock syringe to the Luer adaptor. Inject 3% hot agarose through the black rubber bung to fill the Luer lock syringe using the syringe with needle. Allow 10 to 20 mins for the agarose to cool down and solidify. 
		NOTE: In order to increase the volume capacity of the agarose, the Luer lock syringe is mounted on the Luer adaptor (Figure 4&#160;and&#160;Figure 5). Then, the large electrodes are inserted into the Luer lock syringe. The electrode is capable of providing a stable electrical stimulation for the long-term experiment.</li>
	</ol>
	</li>
</ol>
4. Preparation of mNPCs
<ol>
	<li>Culture the mNPCs&#160;in the complete medium in a 25T cell culture flask at 37 &#176;C in an incubator filled with 5% CO2&#160;atmosphere. Subculture the cells every 3-4 days and perform all experiments with cells that have undergone 3-8 passages from the original source.</li>
	<li>Transfer the cell suspension to a 15 mL conical tube, and spin-down the neurospheres at 100 &#215;&#160;g&#160;for 5 min. Aspirate the supernatant and wash the neurospheres with 1x Dulbecco&#39;s PBS (DPBS). Spin-down the neurospheres at 100 &#215;&#160;g&#160;for 5 min.</li>
	<li>Aspirate the 1x DPBS and then resuspend the neurospheres in the complete medium. Mix thoroughly and gently.</li>
	<li>Add 1 mL of the neurosphere suspension using a 1 mL syringe that connects to the 3-way stopcock of the outlet (Figure 4B-<img alt="Equation 2" src="/files/ftp_upload/22350/22350eq02.jpg" style="margin: auto;" />).</li>
</ol>
5. Setup of the microfluidic system for DC pulse stimulation (Figure 6)
<ol>
	<li>Install the cell-seeded MOE chip onto the transparent indium-tin-oxide (ITO) heater that is fastened on a programmable X-Y-Z motorized stage.&#160;&#160;&#160;&#160;&#160; 
	NOTE: The ITO surface temperature is controlled by a proportional-integral-derivative controller and maintained at 37 &#176;C. A K-type thermocouple is clamped between the chip and the ITO heater to monitor the temperature of the cell culture regions within the chip. The MOE chip is installed on a programmable X-Y-Z motorized stage and is suitable for automatic time-lapse image acquisition at individual channel sections. The fabrication of the ITO heater and the setup of the cell culture heating system have been described previously.</li>
	<li>Infuse the mNPCs by manual pumping into the MOE chip via the medium outlet. Incubate the cell-seeded MOE chip on the 37 &#176;C ITO heater for 4 h.</li>
	<li>After 4 h, pump the complete medium through the MOE chip via the medium inlet at a flow rate of 20 &#181;L/h using a syringe pump.&#160;&#160;&#160;&#160;&#160;&#160; 
	NOTE: The mNPCs are grown and maintained in the chip for an additional 24 h before electric fields (EF) stimulation to allow cell attachment and growth. The waste liquid is collected in an empty 5 mL syringe connected to the 3-way stopcock of the outlet, shown as &#34;waste&#34; in&#160;Figure 6A. The MOE microfluidic system configuration is shown in&#160;Figure 6. This microfluidic system provides a continuous supply of nutrition to the cells. The complete fresh medium is continuously pumped into the MOE chip to maintain a constant pH value. Therefore, the cells can be cultured outside a CO2&#160;incubator.</li>
	<li>Use electrical wires to connect an EF multiplexer to the MOE chip via the Ag/AgCl electrodes on the chip. Connect an EF multiplexer and a function generator to an amplifier to output square-wave DC pulses with a magnitude of 300 mV/mm at a frequency of 100 Hz at 50% duty cycles (50% time-on and 50% time-off) (Figure 6B).
	<ol>
		<li>Connect the electrical wires to the EF multiplexer. Connect the electrical wires to the MOE chip via the Ag/AgCl electrodes.</li>
		<li>Connect the EF multiplexer to the amplifier using electrical wires. Connect the function generator to the amplifier and the digital oscilloscope. 
		NOTE: The EF multiplexer is a circuit that includes the impedance of the culture chamber in the circuit and connects all individual chambers in a parallel electronic network. Each of the three culture chambers is electrically connected in serial to a variable resistor (Vr) and an ammeter (shown as &#181;A in&#160;Figure 6A) in the multiplexer. The electric current through each culture chamber is varied by controlling the Vr, and the current is shown on the corresponding ammeter. The electric field strength in each cell culture region was calculated by Ohm&#39;s Law, I= &#963;EA, where I is the electric current, &#963; (set as 1.38 S&#183;m-1&#160;for DMEM/F12) is the electrical conductivity of the culture medium, E is the electric field, and A is the cross-sectional area of the electrotactic chamber. For the cell culture region dimension shown in&#160;Figure 1, the electric current is ~87 mA and ~44 mA for DC and DC pulse at 50% duty cycle, respectively.</li>
	</ol>
	</li>
	<li>Subject the mNPCs to square DC pulses with a magnitude of 300 mV/mm at the frequency of 100 Hz for 48h. Continuously pump the complete medium at a rate of 10 &#181;L/h to supply adequate nutrition to the cells and to maintain a constant pH value in the medium.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1 mm PMMA substrates (Layers 1-3)</td>
			<td>BHT</td>
			<td>K2R20</td>
			<td>Polymethyl methacrylate (PMMA), http://www.bothharvest.com/zh-tw/product-421076/Optical-PMMA-Non-Coated-BHT-K2Rxx-xx=-thickness-choices.html</td>
		</tr>
		<tr>
			<td>15 mL plastic tube</td>
			<td>Protech Technology Enterprise Co., Ltd</td>
			<td>CT-15-PL-TW</td>
			<td>Conical bottomed tube with cap, assembled, presterilized</td>
		</tr>
		<tr>
			<td>3 mL syringe</td>
			<td>TERUMO</td>
			<td>DVR-3413</td>
			<td>3 mL oral syringes, without needle</td>
		</tr>
		<tr>
			<td>3 mm optical grade PMMA (Layer 5)</td>
			<td>CHI MEI Corporation</td>
			<td>ACRYPOLY PMMA Sheet</td>
			<td>Optical grade PMMA</td>
		</tr>
		<tr>
			<td>3-way stopcock</td>
			<td>NIPRO</td>
			<td>NCN-3L</td>
			<td>Sterile disposable 3-way stopcock</td>
		</tr>
		<tr>
			<td>5 mL syringe</td>
			<td>TERUMO</td>
			<td>DVR-3410</td>
			<td>5 mL oral syringes, without needle</td>
		</tr>
		<tr>
			<td>Adaptor</td>
			<td>Dong Zhong Co., Ltd.</td>
			<td>Customized</td>
			<td>PMMA adaptor</td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Sigma-Aldrich</td>
			<td>A9414</td>
			<td>Agarose, low gelling temperature</td>
		</tr>
		<tr>
			<td>Amplifier</td>
			<td>A.A. Lab Systems Ltd</td>
			<td>A-304</td>
			<td>High voltage amplifier</td>
		</tr>
		<tr>
			<td>AutoCAD software</td>
			<td>Autodesk</td>
			<td>Educational Version</td>
			<td>Drafting</td>
		</tr>
		<tr>
			<td>B-27 supplement</td>
			<td>Gibco</td>
			<td>12587-010</td>
			<td>B-27 supplement (50x), minus vitamin A</td>
		</tr>
		<tr>
			<td>Basic fibroblast growth factor (bFGF)&#160;</td>
			<td>Peprotech</td>
			<td>AF-100-18B</td>
			<td>Also called recombinant human FGF-basic</td>
		</tr>
		<tr>
			<td>Black rubber bung</td>
			<td>TERUMO</td>
			<td>DVR-3413</td>
			<td>From 3 mL oral syringes, without needle</td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA)</td>
			<td>Sigma-Aldrich</td>
			<td>B4287</td>
			<td>Blocking reagent&#160;</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>HSIANGTAI</td>
			<td>CV2060</td>
			<td>Centrifuge</td>
		</tr>
		<tr>
			<td>CO2&#160;laser scriber</td>
			<td>Laser Tools and Technics Corp.&#160;</td>
			<td>ILS-II</td>
			<td>Purchased from http://www.lttcorp.com/index.htm</td>
		</tr>
		<tr>
			<td>Cone connector</td>
			<td>IDEX Health &#38; Science</td>
			<td>F-120X</td>
			<td>One-piece fingertight 10-32 coned, for 1/16&#34; OD natural</td>
		</tr>
		<tr>
			<td>Cone-Luer adaptor</td>
			<td>IDEX Health &#38; Science</td>
			<td>P-659</td>
			<td>Luer Adapter 10-32 Female to Female Luer, PEEK</td>
		</tr>
		<tr>
			<td>Confocal fluorescence microscope</td>
			<td>Leica Microsystems</td>
			<td>TCS SP5</td>
			<td>Leica TCS SP5 user manual, http://www3.unifr.ch/bioimage/wp-content/uploads/2013/10/User-Manual_TCS_SP5_V02_EN.pdf</td>
		</tr>
		<tr>
			<td>Digital camera</td>
			<td>OLYMPUS</td>
			<td>E-330</td>
			<td>Automatic time-lapse image acquisition</td>
		</tr>
		<tr>
			<td>Digital oscilloscope</td>
			<td>Tektronix</td>
			<td>TDS2024</td>
			<td>Measure voltage or current signals over time in an electronic circuit or component to display amplitude and frequency.</td>
		</tr>
		<tr>
			<td>Double-sided tape</td>
			<td>3M&#160;</td>
			<td>PET 8018</td>
			<td>Purchased from http://en.thd.com.tw/</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s modified Eagle&#39;s medium/Ham&#39;s nutrient mixture F-12 (DMEM/F12)</td>
			<td>Gibco</td>
			<td>12400024</td>
			<td>DMEM/F-12, powder, HEPES</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s phosphate-buffered saline (DPBS)</td>
			<td>Gibco</td>
			<td>21600010</td>
			<td>DPBS, powder, no calcium, no magnesium</td>
		</tr>
		<tr>
			<td>EF multiplexer</td>
			<td>Asiatic Sky Co., Ltd.</td>
			<td>Customized</td>
			<td>Monitor and control the electric current in individual channels</td>
		</tr>
		<tr>
			<td>Epidermal growth factor (EGF)</td>
			<td>Peprotech</td>
			<td>AF-100-15</td>
			<td>Also called recombinant human EGF</td>
		</tr>
		<tr>
			<td>Fast-acting cyanoacrylate glue</td>
			<td>3M&#160;</td>
			<td>7004T</td>
			<td>Strength instant adhesive (liquid)</td>
		</tr>
		<tr>
			<td>Flat bottom connector</td>
			<td>IDEX Health &#38; Science</td>
			<td>P-206</td>
			<td>Flangeless male nut Delrin, 1/4-28 flat-bottom, for 1/16&#34; OD blue</td>
		</tr>
		<tr>
			<td>Function generator</td>
			<td>Agilent Technologies</td>
			<td>33120A</td>
			<td>High-performance 15 MHz synthesized&#160;function generator&#160;with built-in arbitrary waveform capability</td>
		</tr>
		<tr>
			<td>Indium&#8211;tin&#8211;oxide (ITO) glass</td>
			<td>Merck</td>
			<td>300739</td>
			<td>For ITO heater</td>
		</tr>
		<tr>
			<td>K-type thermocouple</td>
			<td>Tecpel</td>
			<td>TPK-02A</td>
			<td>Temperature&#160;thermocouples</td>
		</tr>
		<tr>
			<td>Luer adapter</td>
			<td>IDEX Health &#38; Science</td>
			<td>P618-01</td>
			<td>Luer adapter female Luer to 1/4-28 male polypropylene</td>
		</tr>
		<tr>
			<td>Luer lock syringe</td>
			<td>TERUMO</td>
			<td>DVR-3413</td>
			<td>For agar salt bridges</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS)</td>
			<td>Basic Life</td>
			<td>BL2651</td>
			<td>Washing solution</td>
		</tr>
		<tr>
			<td>Poly-L-Lysine (PLL)</td>
			<td>SIGMA</td>
			<td>P4707</td>
			<td>Coating solution</td>
		</tr>
		<tr>
			<td>Precision cover glasses thickness No. 1.5H</td>
			<td>MARIENFELD</td>
			<td>107242</td>
			<td>https://www.marienfeld-superior.com/precision-cover-glasses-thickness-no-1-5h-tol-5-m.html</td>
		</tr>
		<tr>
			<td>Programmable X-Y-Z motorised stage</td>
			<td>Tanlian Inc</td>
			<td>Customized</td>
			<td>Purchased from http://www.tanlian.tw/ndex.files/motort.htm</td>
		</tr>
		<tr>
			<td>Proportional&#8211;integral&#8211;derivative (PID) controller</td>
			<td>Toho Electronics</td>
			<td>TTM-J4-R-AB</td>
			<td>Temperature controller&#160;</td>
		</tr>
		<tr>
			<td>PTFE tube</td>
			<td>Professional Plastics Inc. Taiwan Branch</td>
			<td>Outer diameter 1/16 Inches</td>
			<td>White translucent PTFE tubing</td>
		</tr>
		<tr>
			<td>Syringe pump</td>
			<td>New Era Systems Inc</td>
			<td>NE-1000</td>
			<td>NE-1000 programmable single syringe pump</td>
		</tr>
		<tr>
			<td>TFD4 detergent</td>
			<td>FRANKLAB</td>
			<td>TFD4</td>
			<td>Cover glass cleaner</td>
		</tr>
		<tr>
			<td>Thermal bonder</td>
			<td>Kuan-MIN Tech Co.</td>
			<td>Customized</td>
			<td>Purchased from http://kmtco.com.tw/</td>
		</tr>
		<tr>
			<td>Ultrasonic cleaner</td>
			<td>LEO</td>
			<td>LEO-300S</td>
			<td>Ultrasonic steri-cleaner</td>
		</tr>
		<tr>
			<td>Vacuum chamber</td>
			<td>DENG YNG INSTRUMENTS CO., Ltd.</td>
			<td>DOV-30</td>
			<td>Vacuum drying oven</td>
		</tr>
		<tr>
			<td>White fingertight plug</td>
			<td>IDEX Health &#38; Science</td>
			<td>P-316</td>
			<td>1/4-28 Flat-Bottom, https://www.idex-hs.com/store/fluidics/fluidic-connections/plug-teflonr-pfa-1-4-28-flat-bottom.html</td>
		</tr>
	</tbody>
</table>

Source: Chang, H. et al., <a href="https://app.jove.com/v/61917">Electric-Field-Induced Neural Precursor Cell Differentiation in Microfluidic Devices.</a>&#160;J. Vis. Exp. (2021)
This video demonstrates the use of an extracellular matrix-coated microfluidic chip to differentiate neural stem and progenitor cells or NPCs through electrical stimulation. After seeding and adhering to the coated region, NPCs grow, and the application of a direct current&#160; triggers their differentiation into neurons, astrocytes, and oligodendrocytes.

<img alt="Figure 1" src="/files/ftp_upload/22350/22350_Figure1.jpg" /> 
Figure 1: The detailed configuration of the multichannel optically transparent electrotactic chip.&#160;(A) Exploded view of the MOE chip assembly. The MOE chip consists of PMMA sheets (50 mm x 25 mm x 1 mm), double-sided tape (50 mm x 25 mm x 0.07 mm), adaptors (10 mm x 10 mm x 6 mm), optical grade PMMA sheet (50 mm x 75 mm x 3 mm), double-sided tape (24 mm x 60 mm x 0.07 mm), a...

1. Design and fabrication of the multichannel, optically transparent, electrotactic (MOE) chip

	Draw patterns for individual polymethyl methacrylate ...

Begin with a MOE microfluidic chip connected to an electrical supply.
The chip contains poly-L-lysine-coated culture regions connected with inlet and outlet ports, along with mounted Ag/AgCl electrodes for electrical connections.
Place the microfluidic chip on a heater for optimal growth conditions.
Flow-in neural stem and progenitor cells or NPCs into the coated culture regions via the outlets.&#160;
The cells electrostatically interact with the poly-L-lysine and adhere to the microfluidic device.
Next, introduce a growth medium containing growth factors that facilitate the proliferation of the adhered cells.
Apply an electric current via the Ag/AgCl electrodes for a longer duration with continuous supply of medium for cell viability.
The electrical current excites and activates NPCs, promoting the extension of neuronal processes from the cell bodies.
Additionally, electrical stimulation triggers various intracellular signaling pathways.
These pathways promote NPCs to differentiate specifically into various neural cells, such as neurons, astrocytes, and oligodendrocytes.

17 visualizações. Diferenciação induzida por estimulação elétrica de células-tronco neurais e progenitoras

Vídeo: Diferenciação induzida por estimulação elétrica de células-tronco neurais e progenitoras - Experimento

Rapid Detection of Neurodevelopmental Phenotypes in Human Neural Precursor Cells (NPCs)

Human brain development proceeds through a series of precisely orchestrated processes, with earlier stages distinguished by proliferation, migration, and neurite outgrowth; and later stages characterized by axon/dendrite outgrowth and synapse formation. In neurodevelopmental disorders, often one or more of these processes are disrupted, leading to abnormalities in brain formation and function. With the advent of human induced pluripotent stem cell (hiPSC) technology, researchers now have an abundant supply of human cells that can be differentiated into virtually any cell type, including neurons. These cells can be used to study both normal brain development and disease pathogenesis. A number of protocols using hiPSCs to model neuropsychiatric disease use terminally differentiated neurons or use 3D culture systems termed organoids. While these methods have proven invaluable in studying human disease pathogenesis, there are some drawbacks. Differentiation of hiPSCs into neurons and generation of organoids are lengthy and costly processes that can impact the number of experiments and variables that can be assessed. In addition, while post-mitotic neurons and organoids allow the study of disease-related processes, including dendrite outgrowth and synaptogenesis, they preclude the study of earlier processes like proliferation and migration. In neurodevelopmental disorders, such as autism, abundant genetic and post-mortem evidence indicates defects in early developmental processes. Neural precursor cells (NPCs), a highly proliferative cell population, may be a suitable model in which to ask questions about ontogenetic processes and disease initiation. We now extend methodologies learned from studying development in mouse and rat cortical cultures to human NPCs. The use of NPCs allows us to investigate disease-related phenotypes and define how different variables (e.g., growth factors, drugs) impact developmental processes including proliferation, migration, and differentiation in only a few days. Ultimately, this toolset can be used in a reproducible and high-throughput manner to identify disease-specific mechanisms and phenotypes in neurodevelopmental disorders.

Rapid Detection of Neurodevelopmental Phenotypes in Human Neural Precursor Cells NPCs

Neurodevelopmental processes such as proliferation, migration, and neurite outgrowth are often perturbed in neuropsychiatric diseases. Thus, we present protocols to rapidly and reproducibly assess these neurodevelopmental processes in human iPSC-derived NPCs. These protocols also allow the assessment of the effects of relevant growth factors and therapeutics on NPC development.

Detecção rápida de desenvolvimento neurológico fenótipos nas células precursoras neurais humanas (NPCs)

Human brain development proceeds through a series of precisely orchestrated processes, with earlier stages distinguished by proliferation, migration, and ...

JoVE Journal

Biologia do Desenvolvimento

Compartmentalization of Human Stem Cell-Derived Neurons within Pre-Assembled Plastic Microfluidic Chips

Use of microfluidic devices to compartmentalize cultured neurons has become a standard method in neuroscience. This protocol shows how to use a pre-assembled multi-compartment chip made in a cyclic olefin copolymer (COC) to compartmentalize neurons differentiated from human stem cells. The footprint of these COC chips are the same as a standard microscope slide and are equally compatible with high resolution microscopy. Neurons are differentiated from human neural stem cells (NSCs) into glutamatergic neurons within the chip and maintained for 5 weeks, allowing sufficient time for these neurons to develop synapses and dendritic spines. Further, we demonstrate multiple common experimental procedures using these multi-compartment chips, including viral labeling, establishing microenvironments, axotomy, and immunocytochemistry.

This protocol demonstrates the use of compartmentalized microfluidic chips, injection molded in a cyclic olefin copolymer to cultured neurons differentiated from human stem cells. These chips are preassembled and easier-to-use than traditional compartmentalized poly(dimethylsiloxane) devices. Multiple common experimental paradigms are described here, including viral labeling, fluidic isolation, axotomy, and immunostaining.

Compartimentalização de neurônios derivados de células-tronco humanas dentro de chips de plástico Microfluídico pré-montados

Use of microfluidic devices to compartmentalize cultured neurons has become a standard method in neuroscience. This protocol shows how to use a ...

Neurociência

High-Content Screening Differentiation and Maturation Analysis of Fetal and Adult Neural Stem Cell-Derived Oligodendrocyte Precursor Cell Cultures

The main hurdle in developing drug screening techniques for assessing the efficacy of therapeutic strategies in complex diseases is striking a balance between in vitro simplification and recreating the complex in vivo environment, along with the main aim, shared by all screening strategies, of obtaining robust and reliable data, highly predictive for in vivo translation.
In the field of demyelinating diseases, the majority of drug screening strategies are based on immortalized cell lines or pure cultures of isolated primary oligodendrocyte precursor cells (OPCs) from newborn animals, leading to strong biases due to the lack of age-related differences and of any real pathological condition or complexity.
Here we show the setup of an in vitro system aimed at modeling the physiological differentiation/maturation of neural stem cell (NSC)-derived OPCs, easily manipulated to mimic pathological conditions typical of demyelinating diseases. Moreover, the method includes isolation from fetal and adult brains, giving a system which dynamically differentiates from OPCs to mature oligodendrocytes (OLs) in a spontaneous co-culture which also includes astrocytes. This model physiologically resembles the thyroid hormone-mediated myelination and myelin repair process, allowing the addition of pathological interferents which model disease mechanisms. We show how to mimic the two main components of demyelinating diseases (i.e., hypoxia/ischemia and inflammation), recreating their effect on developmental myelination and adult myelin repair and taking all the cell components of the system into account throughout, while focusing on differentiating OPCs.
This spontaneous mixed model, coupled with cell-based high-content screening technologies, allows the development of a robust and reliable drug screening system for therapeutic strategies aimed at combating the pathological processes involved in demyelination and at inducing remyelination.

We describe the production of mixed cultures of astrocytes and oligodendrocyte precursor cells derived from fetal or adult neural stem cells differentiating into mature oligodendrocytes, and in vitro modeling of noxious stimuli. The coupling with a cell-based high-content screening technique builds a reliable and robust drug screening system.

Análise de diferenciação e maturação de rastreamento de alto conteúdo das culturas celulares precursoras do Oligodendrocyte, derivadas de células-tronco-tronco-tronco-de-alto teor

The main hurdle in developing drug screening techniques for assessing the efficacy of therapeutic strategies in complex diseases is striking a balance ...

Electrical Stimulation-Induced Differentiation of Neural Stem and Progenitor Cells

Transcrição

Electrical Stimulation-Induced Differentiation of Neural Stem and Progenitor Cells