The authors thank Professor Tang K. Tang, Institute of Biomedical Sciences, Academia Sinica, for his assistance in providing mouse neural stem and progenitor cells (mNPCs). The authors also thank Professor Tang K. Tang and Ms. Ying-Shan Lee, for their valuable discussion on the differentiation of mNPCs.

1. Design and fabrication of the 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 laser machine scriber (Figure 1B).
	<ol>
		<li>Switch on the CO2 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. 
	NOTE: The drawing of the PMMA pattern and direct machining of the PMMA sheet were performed according to a previous report17.</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 in a thermal bonder for 30 min at 110 &#176;C to form the flow/electrical stimulation channel assembly (Figure 2C). 
	NOTE: Different batches of commercially obtained PMMA sheet have slightly different glass transition temperature (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. 
	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 thread 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 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/61917/1.png" style="vertical-align: middle;" />).</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/61917/2.png" style="vertical-align: middle;" />).</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 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/61917/1.png" style="vertical-align: middle;" />&#160;and Figure 4B-<img alt="Equation 3" src="/files/ftp_upload/61917/3.png" style="vertical-align: middle;" />).</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/61917/4.png" style="vertical-align: middle;" />).</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 and 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 mNPCs1 in the complete medium in a 25T cell culture flask at 37 &#176;C in an incubator filled with 5% CO2 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; g for 5 min. Aspirate the supernatant, and wash the neurospheres with 1x Dulbecco&#39;s PBS (DPBS). Spin-down the neurospheres at 100 &#215; g 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/61917/2.png" style="vertical-align: middle;" />).</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 ITO heater that is fastened on a programmable X-Y-Z motorized stage. 
	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 previously18,19.</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. 
	NOTE: The mNPCs are grown and maintained in the chip for an additional 24 h before 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 Figure 6A. The MOE microfluidic system configuration is shown in 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 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 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 for DMEM/F1220) 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 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>
6. Immunofluorescence assays of mNPCs after pulsed DC stimulation
NOTE: In this step, all reagent is pumped via the medium inlet using a syringe pump.
<ol>
	<li>After 3, 7, or 14 days in vitro (DIV) culturing after seeding1, wash the cells with 1x PBS at a flow rate of 25 &#181;L/min for 20 min.</li>
	<li>Fix the cells with 4% paraformaldehyde (PFA). Pump 4% PFA into the chip at a flow rate of 25 &#181;L/min for 20 min to replace the 1x PBS. To replace the 4% PFA, wash the cells with 1x PBS at a flow rate of 25 &#181;L/min for 20 min.</li>
	<li>Pump 0.1% Triton X-100 into the chip at a flow rate of 50 &#181;L/min for 6 min to permeabilize the cells. Reduce the flow rate to 50 &#181;L/h for an additional 30 min to react with the cells. To replace the 0.1% Triton X-100, wash the cells with 1x PBS at a flow rate of 50 &#181;L/min for 6 min.</li>
	<li>Block the cells with PBS containing 1% bovine serum albumin (BSA) to reduce nonspecific antibody binding. Pump 1% BSA into the chip at a flow rate of 50 &#181;L/min for 6 min. Reduce the flow rate to 100 &#181;L/h and pump for 1 h.</li>
	<li>Pump the antibodies for double immunostaining into the chip at a flow rate of 50 &#181;L/min for 6 min, and incubate the chip for 18 h at 4 &#176;C. Wash the cells with 1x PBS at a flow rate of 50 &#181;L/min for 15 min.</li>
	<li>Pump the Alexa Fluor-conjugated secondary antibodies into the chip at a flow rate of 50 &#181;L/min for 6 min. Reduce the flow rate to 50 &#181;L/h, and pump the antibodies for 1 h at room temperature in the dark. Wash the cells with 1x PBS at a flow rate of 50 &#181;L/min for 15 min.</li>
	<li>For nuclear staining, pump Hoechst 33342 into the chip at a flow rate of 20 &#181;L/min for 10 min at room temperature in the dark. Wash the cells with 1x PBS at a flow rate of 50 &#181;L/min for 15 min.</li>
	<li>After immunostaining, observe the cells using a confocal fluorescence microscope.</li>
</ol>
7. Image analysis and data processing
<ol>
	<li>Analyze the fluorescent images using software with built-in measurement tools (see the Table of Materials).</li>
	<li>Compare the Hoechst-counterstained nuclei (total number of cells) in the control and treatment groups, and calculate the percentage of cells expressing each phenotypic marker.</li>
</ol>

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The authors have nothing to disclose.

During the fabrication of the MOE chip, the adaptors are attached to the Layer 1 of the MOE chip with fast-acting cyanoacrylate glue. The glue is applied to 4 corners of the adaptors, and then pressure is applied evenly over the adaptors. Excess amount of glue must be avoided to ensure complete polymerization of the glue. Moreover, the completed MOE chip assembly is incubated in a vacuum chamber. This step helps to remove the bubbles between the PMMA layer, the double-sided tape, and the cover glass.
<p class="jove_c...

Neural precursor cells (NPCs, also known as neural stem and progenitor cells) can be as a promising candidate for neurodegenerative therapeutic strategy1. The undifferentiated NPCs have self-renewal capacity, multi-potency, and proliferative ability2,3. A previous study has reported that the extracellular matrix and molecular mediators regulate differentiation of NPC. The epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) promote NPC proliferation, thus maintaining the undifferentiated state4.
Previous studies have reported that electrical stimulation can regulate cell physiologic activities such as division5, migration6,7,8, differentiation1,9,10, and cell death11. Electric fields (EFs) play vital roles in the development and regeneration of the central nervous system development12,13,14. From 2009 to 2019, this laboratory has investigated cellular responses to the application of EF in the microfluidic system1,6,7,8,15,16,17. A multichannel, optically transparent, electrotactic (MOE) chip was designed to be suitable for immunofluorescence staining for confocal microscopy. The chip had high optical transparency and good durability and allowed the simultaneous conduct of three independent stimulation experiments and several immunostained conditions in a single study. The microfluidic system is beneficial in simplifying the overall experimental setup and, in turn, the reagent and sample consumption. This paper describes the development of a microfluidic cell culture system that was used for a long-term cell differentiation study.

<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 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&#8217;s modified Eagle&#8217;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>Goat anti-mouse IgG H&#38;L (Alexa Fluor 488)</td>
			<td>Abcam</td>
			<td>ab150117</td>
			<td>Goat anti-mouse IgG H&#38;L (Alexa Fluor 488) preadsorbed</td>
		</tr>
		<tr>
			<td>Goat anti-rabbit IgG H&#38;L (Alexa Fluor 555)</td>
			<td>Abcam</td>
			<td>ab150086</td>
			<td>Goat polyclonal secondary antibody to rabbit IgG - H&#38;L (Alexa Fluor 555), preadsorbed</td>
		</tr>
		<tr>
			<td>Hoechst 33342</td>
			<td>Invitrogen</td>
			<td>H3570</td>
			<td>Nuclear staining</td>
		</tr>
		<tr>
			<td>ImageJ software</td>
			<td>National Institutes of Health</td>
			<td>1.48v</td>
			<td>Analyze the fluorescent images&#160;</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>Inverted phase contrast microscope</td>
			<td>OLYMPUS</td>
			<td>CKX41</td>
			<td>For cell morphology observation</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>Mouse anti-GFAP</td>
			<td>eBioscience</td>
			<td>14-9892</td>
			<td>Astrocytes marker</td>
		</tr>
		<tr>
			<td>Oligodendrocyte &#160;marker&#160; O4&#160; antibody</td>
			<td>R&#38;D Systems</td>
			<td>MAB1326</td>
			<td>Oligodendrocytes marker</td>
		</tr>
		<tr>
			<td>Paraformaldehyde (PFA)</td>
			<td>Sigma-Aldrich</td>
			<td>P6148</td>
			<td>Fixing&#160;agent</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>Rabbit anti-Tuj1</td>
			<td>Abcam</td>
			<td>ab18207</td>
			<td>Neuron marker</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>Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>T8787</td>
			<td>Permeabilized solution</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>

electric-field-induced neural precursor cell differentiation in microfluidic devices

Physiological electric fields (EF) play vital roles in cell migration, differentiation, division, and death. This paper describes a microfluidic cell culture system that was used for a long-term cell differentiation study using microscopy. The microfluidic system consists of the following major components: an optically transparent electrotactic chip, a transparent indium-tin-oxide (ITO) heater, a culture media-filling pump, an electrical power supply, a high-frequency power amplifier, an EF multiplexer, a programmable X-Y-Z motorized stage, and an inverted phase-contrast microscope equipped with a digital camera. The microfluidic system is beneficial in simplifying the overall experimental setup and, in turn, the reagent and sample consumption. This work involves the differentiation of neural stem and progenitor cells (NPCs) induced by direct current (DC) pulse stimulation. In the stem cell maintenance medium, the mouse NPCs (mNPCs) differentiated into neurons, astrocytes, and oligodendrocytes after the DC pulse stimulation. The results suggest that simple DC pulse treatment could control the fate of mNPCs and could be used to develop therapeutic strategies for nervous system disorders. The system can be used for cell culture in multiple channels, for long-term EF stimulation, for cell morphological observation, and for automatic time-lapse image acquisition. This microfluidic system not only shortens the required experimental time, but also increases the accuracy of control on the microenvironment.

The detailed configuration of the MOE chip is shown in Figure 1. The microfluidic chip provides a beneficial approach for reducing the experimental setup size, sample volume, and reagent volume. The MOE chip was designed to perform three independent EF stimulation experiments and several immunostaining conditions simultaneously in a single study (Figure 3). In addition, the MOE chip, which has a high optical transparency is suita...

Watch this Scientific Journal Video about Electric-Field-Induced Neural Precursor Cell Differentiation in Microfluidic Devices at JoVE.com

Electric-Field-Induced Neural Precursor Cell Differentiation in Microfluidic Devices

In this study, we present a protocol for the differentiation of neural stem and progenitor cells (NPCs) solely induced by direct current (DC) pulse stimulation in a microfluidic system.

Physiological electric fields (EF) play vital roles in cell migration, differentiation, division, and death. This paper describes a microfluidic cell ...

electric-field-induced-neural-precursor-cell-differentiation

Research

JoVE Journal

Bioengineering

3.5K Views.  Academia Sinica. In this study, we present a protocol for the differentiation of neural stem and progenitor cells (NPCs) solely induced by direct current (DC) pulse stimulation in a microfluidic system.

Video: Electric-Field-Induced Neural Precursor Cell Differentiation in Microfluidic Devices

Separating Beads and Cells in Multi-channel Microfluidic Devices Using Dielectrophoresis and Laminar Flow

Microfluidic devices have advanced cell studies by providing a dynamic fluidic environment on the scale of the cell for studying, manipulating, sorting and counting cells. However, manipulating the cell within the fluidic domain remains a challenge and requires complicated fabrication protocols for forming valves and electrodes, or demands specialty equipment like optical tweezers. Here, we demonstrate that conventional printed circuit boards (PCB) can be used for the non-contact manipulation of cells by employing dielectrophoresis (DEP) for bead and cell manipulation in laminar flow fields for bioactuation, and for cell and bead separation in multichannel microfluidic devices. First, we present the protocol for assembling the DEP electrodes and microfluidic devices, and preparing the cells for DEP. Then, we characterize the DEP operation with polystyrene beads. Lastly, we show representative results of bead and cell separation in a multichannel microfluidic device. In summary, DEP is an effective method for manipulating particles (beads or cells) within microfluidic devices.

Dielectrophoresis (DEP) is an effective method to manipulate cells. Printed circuit boards (PCB) can provide inexpensive, reusable and effective electrodes for contact-free cell manipulation within microfluidic devices. By combining PDMS-based microfluidic channels with coverslips on PCBs, we demonstrate bead and cell manipulation and separation within multichannel microfluidic devices.

Microfluidic devices have advanced cell studies by providing a dynamic fluidic environment on the scale of the cell for studying, manipulating, sorting ...

Simple Microfluidic Devices for in vivo Imaging of C. elegans, Drosophila and Zebrafish

Micro fabricated fluidic devices provide an accessible micro-environment for in vivo studies on small organisms. Simple fabrication processes are available for microfluidic devices using soft lithography techniques 1-3. Microfluidic devices have been used for sub-cellular imaging 4,5, in vivo laser microsurgery 2,6 and cellular imaging 4,7. In vivo imaging requires immobilization of organisms. This has been achieved using suction 5,8, tapered channels 6,7,9, deformable membranes 2-4,10, suction with additional cooling 5, anesthetic gas 11, temperature sensitive gels 12, cyanoacrylate glue 13 and anesthetics such as levamisole 14,15. Commonly used anesthetics influence synaptic transmission 16,17 and are known to have detrimental effects on sub-cellular neuronal transport 4. In this study we demonstrate a membrane based poly-dimethyl-siloxane (PDMS) device that allows anesthetic free immobilization of intact genetic model organisms such as Caenorhabditis elegans (C. elegans), Drosophila larvae and zebrafish larvae. These model organisms are suitable for in vivo studies in microfluidic devices because of their small diameters and optically transparent or translucent bodies. Body diameters range from ~10 &mu;m to ~800 &mu;m for early larval stages of C. elegans and zebrafish larvae and require microfluidic devices of different sizes to achieve complete immobilization for high resolution time-lapse imaging. These organisms are immobilized using pressure applied by compressed nitrogen gas through a liquid column and imaged using an inverted microscope. Animals released from the trap return to normal locomotion within 10 min.
We demonstrate four applications of time-lapse imaging in C. elegans namely, imaging mitochondrial transport in neurons, pre-synaptic vesicle transport in a transport-defective mutant, glutamate receptor transport and Q neuroblast cell division. Data obtained from such movies show that microfluidic immobilization is a useful and accurate means of acquiring in vivo data of cellular and sub-cellular events when compared to anesthetized animals (Figure 1J and 3C-F 4).
Device dimensions were altered to allow time-lapse imaging of different stages of C. elegans, first instar Drosophila larvae and zebrafish larvae. Transport of vesicles marked with synaptotagmin tagged with GFP (syt.eGFP) in sensory neurons shows directed motion of synaptic vesicle markers expressed in cholinergic sensory neurons in intact first instar Drosophila larvae. A similar device has been used to carry out time-lapse imaging of heartbeat in ~30 hr post fertilization (hpf) zebrafish larvae. These data show that the simple devices we have developed can be applied to a variety of model systems to study several cell biological and developmental phenomena in vivo.

Simple Microfluidic Devices for in vivo Imaging of C. elegans, Drosophila and Zebrafish

A simple microfluidic device has been developed to perform anesthetic free in vivo imaging of C. elegans, intact Drosophila larvae and zebrafish larvae. The device utilizes a deformable PDMS membrane to immobilize these model organisms in order to perform time lapse imaging of numerous processes such as heart beat, cell division and sub-cellular neuronal transport. We demonstrate the use of this device and show examples of different types of data collected from different model systems.

Micro fabricated fluidic devices provide an accessible micro-environment for in vivo studies on small organisms. Simple fabrication processes are ...

Detection of Exosomal Biomarker by Electric Field-induced Release and Measurement (EFIRM)

Exosomes are microvesicular structures that play a mediating role in intercellular communication. It is of interest to study the internal cargo of exosomes to determine if they carry disease discriminatory biomarkers. For performing exosomal analysis, it is necessary to develop a method for extracting and analyzing exosomes from target biofluids without damaging the internal content.
Electric field-induced release and measurement (EFIRM) is a method for specifically extracting exosomes from biofluids, unloading their cargo, and testing their internal RNA/protein content. Using an anti-human CD63 specific antibody magnetic microparticle, exosomes are first precipitated from biofluids. Following extraction, low-voltage electric cyclic square waves (CSW) are applied to disrupt the vesicular membrane and cause cargo unloading. The content of the exosome is hybridized to DNA primers or antibodies immobilized on an electrode surface for quantification of molecular content.
The EFIRM method is advantageous for extraction of exosomes and unloading cargo for analysis without lysis buffer. This method is capable of performing specific detection of both RNA and protein biomarker targets in the exosome. EFIRM extracts exosomes specifically based on their surface markers as opposed to size-based techniques.
Transmission electron microscopy (TEM) and assay demonstrate the functionality of the method for exosome capture and analysis. The EFIRM method was applied to exosomal analysis of 9 mice injected with human lung cancer H640 cells (a cell line transfected to express the exosome marker human CD63-GFP) in order to test their exosome profile against 11 mice receiving saline controls. Elevated levels of exosomal biomarkers (reference gene GAPDH and protein surface marker human CD63-GFP) were found for the H640 injected mice in both serum and saliva samples. Furthermore, saliva and serum samples were demonstrated to have linearity (R = 0.79). These results are suggestive for the viability of salivary exosome biomarkers for detection of distal diseases.

Detection of Exosomal Biomarker by Electric Field-induced Release and Measurement EFIRM

Exosomes are microvesicular structures found within biofluids that potentially carry important disease discriminatory biomarkers. Here, a novel method is used to specifically extract exosomes and rapidly test the exosomal cargo for both RNA/protein targets following the disruption of exosomes using non-uniform electric cyclic square waves.

Exosomes are microvesicular structures that play a mediating role in intercellular communication. It is of interest to study the internal cargo of ...

Layer-by-layer Collagen Deposition in Microfluidic Devices for Microtissue Stabilization

Although microfluidics provides exquisite control of the cellular microenvironment, culturing cells within microfluidic devices can be challenging. 3D culture of cells in collagen type I gels helps to stabilize cell morphology and function, which is necessary for creating microfluidic tissue models in microdevices. Translating traditional 3D culture techniques for tissue culture plates to microfluidic devices is often difficult because of the limited channel dimensions. In this method, we describe a technique for modifying native type I collagen to generate polycationic and polyanionic collagen solutions that can be used with layer-by-layer deposition to create ultrathin collagen assemblies on top of cells cultured in microfluidic devices. These thin collagen layers stabilize cell morphology and function, as shown using primary hepatocytes as an example cell, allowing for the long term culture of microtissues in microfluidic devices.

The creation of functional microtissues within microfluidic devices requires the stabilization of cell phenotypes by adapting traditional cell culture techniques to the limited spatial dimensions in microdevices. Modification of collagen allows the layer-by-layer deposition of ultrathin collagen assemblies that can stabilize primary cells, such as hepatocytes, as microfluidic tissue models.

Although microfluidics provides exquisite control of the cellular microenvironment, culturing cells within microfluidic devices can be challenging. 3D ...

Using Adhesive Patterning to Construct 3D Paper Microfluidic Devices

We demonstrate the use of patterned aerosol adhesives to construct both planar and nonplanar 3D paper microfluidic devices. By spraying an aerosol adhesive through a metal stencil, the overall amount of adhesive used in assembling paper microfluidic devices can be significantly reduced. We show on a simple 4-layer planar paper microfluidic device that the optimal adhesive application technique and device construction style depends heavily on desired performance characteristics. By moderately increasing the overall area of a device, it is possible to dramatically decrease the wicking time and increase device success rates while also reducing the amount of adhesive required to keep the device together. Such adhesive application also causes the adhesive to form semi-permanent bonds instead of permanent bonds between paper layers, enabling single-use devices to be non-destructively disassembled after use. Nonplanar 3D origami devices also benefit from the semi-permanent bonds during folding, as it reduces the likelihood that unrelated faces may accidently stick together. Like planar devices, nonplanar structures see reduced wicking times with patterned adhesive application vs uniformly applied adhesive.

We demonstrate the use of patterned aerosol adhesives to construct 3D paper microfluidic devices. This method of adhesive application forms semi-permanent bonds between layers, enabling single-use devices to be non-destructively disassembled after use and to ease folding complex nonplanar structures.

We demonstrate the use of patterned aerosol adhesives to construct both planar and nonplanar 3D paper microfluidic devices. By spraying an aerosol ...

Designing Microfluidic Devices for Studying Cellular Responses Under Single or Coexisting Chemical/Electrical/Shear Stress Stimuli

Microfluidic devices are capable of creating a precise and controllable cellular micro-environment of pH, temperature, salt concentration, and other physical or chemical stimuli. They have been commonly used for in vitro cell studies by providing in vivo like surroundings. Especially, how cells response to chemical gradients, electrical fields, and shear stresses has drawn many interests since these phenomena are important in understanding cellular properties and functions. These microfluidic chips can be made of glass substrates, silicon wafers, polydimethylsiloxane (PDMS) polymers, polymethylmethacrylate (PMMA) substrates, or polyethyleneterephthalate (PET) substrates. Out of these materials, PMMA substrates are cheap and can be easily processed using laser ablation and writing. Although a few microfluidic devices have been designed and fabricated for generating multiple, coexisting chemical and electrical stimuli, none of them was considered efficient enough in reducing experimental repeats, particular for screening purposes. In this report, we describe our design and fabrication of two PMMA-based microfluidic chips for investigating cellular responses, in the production of reactive oxygen species and the migration, under single or coexisting chemical/electrical/shear stress stimuli. The first chip generates five relative concentrations of 0, 1/8, 1/2, 7/8, and 1 in the culture regions, together with a shear stress gradient produced inside each of these areas. The second chip generates the same relative concentrations, but with five different electric field strengths created within each culture area. These devices not only provide cells with a precise, controllable micro-environment but also greatly increase the experimental throughput.

Micro-fabricated devices integrated with fluidic components provide an in vitro platform for cell studies mimicking the in vivo micro-environment. We developed polymethylmethacrylate-based microfluidic chips for studying cellular responses under single or coexisting chemical/electrical/shear stress stimuli.

Microfluidic devices are capable of creating a precise and controllable cellular micro-environment of pH, temperature, salt concentration, and other ...

Automated Robotic Liquid Handling Assembly of Modular DNA Devices

Recent advances in modular DNA assembly techniques have enabled synthetic biologists to test significantly more of the available &#34;design space&#34; represented by &#34;devices&#34; created as combinations of individual genetic components. However, manual assembly of such large numbers of devices is time-intensive, error-prone, and costly. The increasing sophistication and scale of synthetic biology research necessitates an efficient, reproducible way to accommodate large-scale, complex, and high throughput device construction.
Here, a DNA assembly protocol using the Type-IIS restriction endonuclease based Modular Cloning (MoClo) technique is automated on two liquid-handling robotic platforms. Automated liquid-handling robots require careful, often times tedious optimization of pipetting parameters for liquids of different viscosities (e.g. enzymes, DNA, water, buffers), as well as explicit programming to ensure correct aspiration and dispensing of DNA parts and reagents. This makes manual script writing for complex assemblies just as problematic as manual DNA assembly, and necessitates a software tool that can automate script generation. To this end, we have developed a web-based software tool, http://mocloassembly.com, for generating combinatorial DNA device libraries from basic DNA parts uploaded as Genbank files. We provide access to the tool, and an export file from our liquid handler software which includes optimized liquid classes, labware parameters, and deck layout. All DNA parts used are available through Addgene, and their digital maps can be accessed via the Boston University BDC ICE Registry. Together, these elements provide a foundation for other organizations to automate modular cloning experiments and similar protocols.
The automated DNA assembly workflow presented here enables the repeatable, automated, high-throughput production of DNA devices, and reduces the risk of human error arising from repetitive manual pipetting. Sequencing data show the automated DNA assembly reactions generated from this workflow are ~95% correct and require as little as 4% as much hands-on time, compared to manual reaction preparation.

Here, an automated workflow to perform modular DNA &#34;device&#34; assembly using a modular cloning DNA assembly method on liquid-handling robots is presented. The protocol uses an intuitive software tool for generating liquid handler picklists for combinatorial DNA device library generation, which we demonstrate using two liquid handling platforms.

Recent advances in modular DNA assembly techniques have enabled synthetic biologists to test significantly more of the available &#34;design space&#34; ...

Multi-step Variable Height Photolithography for Valved Multilayer Microfluidic Devices

Microfluidic systems have enabled powerful new approaches to high-throughput biochemical and biological analysis. However, there remains a barrier to entry for non-specialists who would benefit greatly from the ability to develop their own microfluidic devices to address research questions. Particularly lacking has been the open dissemination of protocols related to photolithography, a key step in the development of a replica mold for the manufacture of polydimethylsiloxane (PDMS) devices. While the fabrication of single height silicon masters has been explored extensively in literature, fabrication steps for more complicated photolithography features necessary for many interesting device functionalities (such as feature rounding to make valve structures, multi-height single-mold patterning, or high aspect ratio definition) are often not explicitly outlined. 
Here, we provide a complete protocol for making multilayer microfluidic devices with valves and complex multi-height geometries, tunable for any application. These fabrication procedures are presented in the context of a microfluidic hydrogel bead synthesizer and demonstrate the production of droplets containing polyethylene glycol (PEG diacrylate) and a photoinitiator that can be polymerized into solid beads. This protocol and accompanying discussion provide a foundation of design principles and fabrication methods that enables development of a wide variety of microfluidic devices. The details included here should allow non-specialists to design and fabricate novel devices, thereby bringing a host of recently developed technologies to their most exciting applications in biological laboratories.

Multilayer microfluidic devices often involve the fabrication of master molds with complex geometries for functionality. This article presents a complete protocol for multi-step photolithography with valves and variable height features tunable to any application. As a demonstration, we fabricate a microfluidic droplet generator capable of producing hydrogel beads.

Microfluidic systems have enabled powerful new approaches to high-throughput biochemical and biological analysis. However, there remains a barrier to ...

Fabrication of Three-dimensional Paper-based Microfluidic Devices for Immunoassays

Paper wicks fluids autonomously due to capillary action. By patterning paper with hydrophobic barriers, the transport of fluids can be controlled and directed within a layer of paper. Moreover, stacking multiple layers of patterned paper creates sophisticated three-dimensional microfluidic networks that can support the development of analytical and bioanalytical assays. Paper-based microfluidic devices are inexpensive, portable, easy to use, and require no external equipment to operate. As a result, they hold great promise as a platform for point-of-care diagnostics. In order to properly evaluate the utility and analytical performance of paper-based devices, suitable methods must be developed to ensure their manufacture is reproducible and at a scale that is appropriate for laboratory settings. In this manuscript, a method to fabricate a general device architecture that can be used for paper-based immunoassays is described. We use a form of additive manufacturing (multi-layer lamination) to prepare devices that comprise multiple layers of patterned paper and patterned adhesive. In addition to demonstrating the proper use of these three-dimensional paper-based microfluidic devices with an immunoassay for human chorionic gonadotropin (hCG), errors in the manufacturing process that may result in device failures are discussed. We expect this approach to manufacturing paper-based devices will find broad utility in the development of analytical applications designed specifically for limited-resource settings.

We detail a method to fabricate three-dimensional paper-based microfluidic devices for use in the development of immunoassays. Our approach to device assembly is a type of multilayer, additive manufacturing. We demonstrate a sandwich immunoassay to provide representative results for these types of paper-based devices.

Paper wicks fluids autonomously due to capillary action. By patterning paper with hydrophobic barriers, the transport of fluids can be controlled and ...

Polydimethylsiloxane-polycarbonate Microfluidic Devices for Cell Migration Studies Under Perpendicular Chemical and Oxygen Gradients

This paper reports a microfluidic device made of polydimethylsiloxane (PDMS) with an embedded polycarbonate (PC) thin film to study cell migration under combinations of chemical and oxygen gradients. Both chemical and oxygen gradients can greatly affect cell migration in vivo; however, due to technical limitations, very little research has been performed to investigate their effects in vitro. The device developed in this research takes advantage of a series of serpentine-shaped channels to generate the desired chemical gradients and exploits a spatially confined chemical reaction method for oxygen gradient generation. The directions of the chemical and oxygen gradients are perpendicular to each other to enable straightforward migration result interpretation. In order to efficiently generate the oxygen gradients with minimal chemical consumption, the embedded PC thin film is utilized as a gas diffusion barrier. The developed microfluidic device can be actuated by syringe pumps and placed into a conventional cell incubator during cell migration experiments to allow for setup simplification and optimized cell culture conditions. In cell experiments, we used the device to study migrations of adenocarcinomic human alveolar basal epithelial cells, A549, under combinations of chemokine (stromal cell-derived factor, SDF-1&#945;) and oxygen gradients. The experimental results show that the device can stably generate perpendicular chemokine and oxygen gradients and is compatible with cells. The migration study results indicate that oxygen gradients may play an essential role in guiding cell migration, and cellular behavior under combinations of gradients cannot be predicted from those under single gradients. The device provides a powerful and practical tool for researchers to study interactions between chemical and oxygen gradients in cell culture, which can promote better cell migration studies in more in vivo-like microenvironments.

The control of chemical and oxygen gradients is essential for cell cultures. This paper reports a polydimethylsiloxane-polycarbonate (PDMS-PC) microfluidic device capable of reliably generating combinations of chemical and oxygen gradients for cell migration studies, which can be practically utilized in biological labs without sophisticated instrumentation.

This paper reports a microfluidic device made of polydimethylsiloxane (PDMS) with an embedded polycarbonate (PC) thin film to study cell migration under ...