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>

<ol><li>Chang, H. F., Lee, Y. S., Tang, T. K., Cheng, J. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pulsed+DC+electric+field-induced+differentiation+of+cortical+neural+precursor+cells%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Pulsed DC electric field-induced differentiation of cortical neural precursor cells.</a> PLoS One. 11 (6), e0158133(2016).</li>
<li>Li, S., Li, H., Wang, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Orientation+of+spiral+ganglion+neurite+extension+in+electrical+fields+of+charge-balanced+biphasic+pulses+and+direct+current+in+vitro%5BTitle%5D)+AND+%22Hearing+research%22%5BJournal%5D)">Orientation of spiral ganglion neurite extension in electrical fields of charge-balanced biphasic pulses and direct current in vitro.</a> Hearing research. 267 (1-2), 111-118 (2010).</li>
<li>Rajnicek, A. M., Robinson, K. R., McCaig, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+direction+of+neurite+growth+in+a+weak+DC+electric+field+depends+on+the+substratum%3A+contributions+of+adhesivity+and+net+surface+charge%5BTitle%5D)+AND+%22Developmental+biology%22%5BJournal%5D)">The direction of neurite growth in a weak DC electric field depends on the substratum: contributions of adhesivity and net surface charge.</a> Developmental biology. 203 (2), 412-423 (1998).</li>
<li>Kim, Y. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Differential+regulation+of+proliferation+and+differentiation+in+neural+precursor+cells+by+the+Jak+pathway%5BTitle%5D)+AND+%22Stem+Cells%22%5BJournal%5D)">Differential regulation of proliferation and differentiation in neural precursor cells by the Jak pathway.</a> Stem Cells. 28 (10), 1816-1828 (2010).</li>
<li>Cunha, F., Rajnicek, A. M., McCaig, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Electrical+stimulation+directs+migration%2C+enhances+and+orients+cell+division+and+upregulates+the+chemokine+receptors+CXCR4+and+CXCR2+in+endothelial+cells%5BTitle%5D)+AND+%22Journal+of+Vascular+Research%22%5BJournal%5D)">Electrical stimulation directs migration, enhances and orients cell division and upregulates the chemokine receptors CXCR4 and CXCR2 in endothelial cells.</a> Journal of Vascular Research. 56 (1), 39-53 (2019).</li>
<li>Chang, H. F., Cheng, H. T., Chen, H. Y., Yeung, W. K., Cheng, J. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Doxycycline+inhibits+electric+field-induced+migration+of+non-small+cell+lung+cancer+%28NSCLC%29+cells%5BTitle%5D)+AND+%22Scientific+Reports%22%5BJournal%5D)">Doxycycline inhibits electric field-induced migration of non-small cell lung cancer (NSCLC) cells.</a> Scientific Reports. 9 (1), 8094(2019).</li>
<li>Tsai, H. F., Peng, S. W., Wu, C. Y., Chang, H. F., Cheng, J. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Electrotaxis+of+oral+squamous+cell+carcinoma+cells+in+a+multiple-electric-field+chip+with+uniform+flow+field%5BTitle%5D)+AND+%22Biomicrofluidics%22%5BJournal%5D)">Electrotaxis of oral squamous cell carcinoma cells in a multiple-electric-field chip with uniform flow field.</a> Biomicrofluidics. 6 (3), 34116(2012).</li>
<li>Huang, C. W., Cheng, J. Y., Yen, M. H., Young, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Electrotaxis+of+lung+cancer+cells+in+a+multiple-electric-field+chip%5BTitle%5D)+AND+%22Biosensors+and+Bioelectronics%22%5BJournal%5D)">Electrotaxis of lung cancer cells in a multiple-electric-field chip.</a> Biosensors and Bioelectronics. 24 (12), 3510-3516 (2009).</li>
<li>Jing, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Study+of+electrical+stimulation+with+different+electric-field+intensities+in+the+regulation+of+the+differentiation+of+PC12+cells%5BTitle%5D)+AND+%22American+Chemical+Society+Chemical+Neuroscience%22%5BJournal%5D)">Study of electrical stimulation with different electric-field intensities in the regulation of the differentiation of PC12 cells.</a> American Chemical Society Chemical Neuroscience. 10 (1), 348-357 (2019).</li>
<li>Guo, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Self-powered+electrical+stimulation+for+enhancing+neural+differentiation+of+mesenchymal+stem+cells+on+graphene-poly%283%2C4-ethylenedioxythiophene%29+hybrid+microfibers%5BTitle%5D)+AND+%22American+Chemical+Society+Nano%22%5BJournal%5D)">Self-powered electrical stimulation for enhancing neural differentiation of mesenchymal stem cells on graphene-poly(3,4-ethylenedioxythiophene) hybrid microfibers.</a> American Chemical Society Nano. 10 (5), 5086-5095 (2016).</li>
<li>Manabe, M., Mie, M., Yanagida, Y., Aizawa, M., Kobatake, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Combined+effect+of+electrical+stimulation+and+cisplatin+in+HeLa+cell+death%5BTitle%5D)+AND+%22Biotechnology+and+Bioengineering%22%5BJournal%5D)">Combined effect of electrical stimulation and cisplatin in HeLa cell death.</a> Biotechnology and Bioengineering. 86 (6), 661-666 (2004).</li>
<li>Liu, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Protective+effect+of+moderate+exogenous+electric+field+stimulation+on+activating+netrin-1%2FDCC+expression+against+mechanical+stretch-induced+injury+in+spinal+cord+neurons%5BTitle%5D)+AND+%22Neurotoxicity+Research%22%5BJournal%5D)">Protective effect of moderate exogenous electric field stimulation on activating netrin-1/DCC expression against mechanical stretch-induced injury in spinal cord neurons.</a> Neurotoxicity Research. 34 (2), 285-294 (2018).</li>
<li>Haan, N., Song, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Therapeutic+application+of+electric+fields+in+the+injured+nervous+system%5BTitle%5D)+AND+%22Advances+in+Wound+Care%22%5BJournal%5D)">Therapeutic application of electric fields in the injured nervous system.</a> Advances in Wound Care. 3 (2), 156-165 (2014).</li>
<li>Ariza, C. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+influence+of+electric+fields+on+hippocampal+neural+progenitor+cells%5BTitle%5D)+AND+%22Stem+Cell+Reviews+and+Reports%22%5BJournal%5D)">The influence of electric fields on hippocampal neural progenitor cells.</a> Stem Cell Reviews and Reports. 6 (4), 585-600 (2010).</li>
<li>Huang, C. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Gene+expression+of+human+lung+cancer+cell+line+CL1-5+in+response+to+a+direct+current+electric+field%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Gene expression of human lung cancer cell line CL1-5 in response to a direct current electric field.</a> PLoS One. 6 (10), e25928(2011).</li>
<li>Sun, Y. S., Peng, S. W., Lin, K. H., Cheng, J. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Electrotaxis+of+lung+cancer+cells+in+ordered+three-dimensional+scaffolds%5BTitle%5D)+AND+%22Biomicrofluidics%22%5BJournal%5D)">Electrotaxis of lung cancer cells in ordered three-dimensional scaffolds.</a> Biomicrofluidics. 6 (1), 14102-14114 (2012).</li>
<li>Hou, H. S., Chang, H. F., Cheng, J. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Electrotaxis+studies+of+lung+cancer+cells+using+a+multichannel+dual-electric-field+microfluidic+chip%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">Electrotaxis studies of lung cancer cells using a multichannel dual-electric-field microfluidic chip.</a> Journal of Visualized Experiments. 106, e53340(2015).</li>
<li>Cheng, J. Y., Yen, M. H., Hsu, W. C., Jhang, J. H., Young, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((ITO+patterning+by+a+low+power+Q-switched+green+laser+and+its+use+in+the+fabrication+of+a+transparent+flow+meter%5BTitle%5D)+AND+%22Journal+of+Micromechanics+and+Microengineering%22%5BJournal%5D)">ITO patterning by a low power Q-switched green laser and its use in the fabrication of a transparent flow meter.</a> Journal of Micromechanics and Microengineering. 17 (11), 2316-2323 (2007).</li>
<li>Cheng, J. Y., Yen, M. H., Kuo, C. T., Young, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+transparent+cell-culture+microchamber+with+a+variably+controlled+concentration+gradient+generator+and+flow+field+rectifier%5BTitle%5D)+AND+%22Biomicrofluidics%22%5BJournal%5D)">A transparent cell-culture microchamber with a variably controlled concentration gradient generator and flow field rectifier.</a> Biomicrofluidics. 2 (2), 24105(2008).</li>
<li>Fuhr, G., Shirley, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cell+handling+and+characterization+using+micron+and+submicron+electrode+arrays%3A+state+of+the+art+and+perspectives+of+semiconductor+microtools%5BTitle%5D)+AND+%22Journal+of+Micromechanics+and+Microengineering%22%5BJournal%5D)">Cell handling and characterization using micron and submicron electrode arrays: state of the art and perspectives of semiconductor microtools.</a> Journal of Micromechanics and Microengineering. 5 (2), 77-85 (1995).</li>
<li>AChang, K. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Biphasic+electrical+currents+stimulation+promotes+both+proliferation+and+differentiation+of+fetal+neural+stem+cells%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Biphasic electrical currents stimulation promotes both proliferation and differentiation of fetal neural stem cells.</a> PLoS One. 6 (4), e18738(2011).</li>
<li>Lim, J. H., McCullen, S. D., Piedrahita, J. A., Loboa, E. G., Olby, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Alternating+current+electric+fields+of+varying+frequencies%3A+effects+on+proliferation+and+differentiation+of+porcine+neural+progenitor+cells%5BTitle%5D)+AND+%22Cell+Reprogram%22%5BJournal%5D)">Alternating current electric fields of varying frequencies: effects on proliferation and differentiation of porcine neural progenitor cells.</a> Cell Reprogram. 15 (5), 405-412 (2013).</li>
</ol>

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.
The choice of the electrode material is based on the fact that chloride ions, which are abundantly present in the medium, are the electrolytic products flowing through the cell culture region. During the EF stimulation experiment, the pH around the electrodes remained constant. A simpler configuration using platinum (Pt) as the electrode material electrolyzes water and generates hydrogen ions (H+) and hydroxide ions (OH-) at the positive electrode and the negative electrode, respectively, inducing pH changes in the culture region. Avoiding the use of Pt electrodes circumvents the problem of pH changes during the EF stimulation experiment.
The hot agarose and bubble-free agarose are essential during the preparation of the salt bridge network. The hot agarose has high fluidity and can be easily injected into the salt bridge network. Connect the Luer lock syringe to the Luer adaptor after injecting the 3% hot agarose into the Luer adaptor. During this step, the agarose will be pushed up into the Luer lock syringe so that a bubble-free firm connection of the salt bridge network can be achieved. Bubbles in the salt bridges increase the electrical resistance and hence, the anticipated electric current cannot be reached. After the agarose injection, it is important to wait for the agarose to cool down and solidify at room temperature for 10-20 min to prevent the formation of solidified agarose debris in the cell culture region.
The MOE chip is placed onto an ITO heater that is locked on a programmable X-Y-Z motorized stage. The entire system is built onto an inverted phase-contrast microscope equipped with a digital camera to monitor cell differentiation within the cell culture regions in the chip. It is convenient to observe the cell morphology and acquisition of the automatic time-lapse images in the MOE microfluidic system outside an incubator. This microfluidic system not only shortens the required experimental time, but also increases the accuracy of control on the microenvironment.
The mNPC cells grow as a suspension in culture media. However, mNPCs adhering to the PLL-coated plate in the MOE chip are critical for differentiation. Neurospheres formed by 30-40 cells are preferred for initiating mNPC differentiation. Overgrowth of mNPCs will impair cell survival during the differentiation process. Furthermore, after the pulsed DC stimulation, the immunofluorescence staining experimental can be affected by the flow rate. Hence, use several flow rates for different steps to avoid detaching cells during the wash.
In this study, a limitation of this technique is that the MOE chip cannot be reused because of the difficulty in thorough cleaning of the chip. However, the MOE chip can be placed under a phase-contrast microscope or a scanning confocal microscope directly. The water-tight design of the reported microfluidic system ensures that buffer/medium evaporation does not occur, maintaining the accurate concentration of the buffer/medium and the corresponding electrical properties. By reducing reagent volumes and the corresponding operation time, the MOE microfluidic system provides an efficient approach for studying cell differentiation.
A previous study has shown that EGF and bFGF promote NPC survival, expansion, and maintenance in the undifferentiated state4. In this study, the DC pulses induced the differentiation of the mNPCs in the stem cell maintenance medium that contained EGF and bFGF. Previous studies have reported that EF promotes differentiation of NPCs into neurons and/or astrocytes in differentiation medium without EGF and bFGF14,21,22. These results show that the mNPCs differentiated into neurons, astrocytes, and oligodendrocytes after the DC pulse stimulation. They also suggest that simple DC pulse treatment could control the fate of NPCs. With further optimization on the stimulation time, EF strength, or duty cycle, DC pulses may be applied to manipulate NPC differentiation and may be used for the development of therapeutic strategies that employ NPCs to treat nervous system disorders.

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><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)&amp;nbsp;</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&amp;nbsp;</td></tr><tr><td>Centrifuge</td><td>HSIANGTAI</td><td>CV2060</td><td>Centrifuge</td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; laser scriber</td><td>Laser Tools and Technics Corp.&amp;nbsp;</td><td>ILS-II</td><td>Purchased from http://www.lttcorp.com/index.htm</td></tr><tr><td>Cone connector</td><td>IDEX Health &amp;amp; Science</td><td>F-120X</td><td>One-piece fingertight 10-32 coned, for 1/16&quot; OD natural</td></tr><tr><td>Cone-Luer adaptor</td><td>IDEX Health &amp;amp; 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&amp;nbsp;</td><td>PET 8018</td><td>Purchased from http://en.thd.com.tw/</td></tr><tr><td>Dulbecco&amp;rsquo;s modified Eagle&amp;rsquo;s medium/Ham&#039;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&#039;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&amp;nbsp;</td><td>7004T</td><td>Strength instant adhesive (liquid)</td></tr><tr><td>Flat bottom connector</td><td>IDEX Health &amp;amp; Science</td><td>P-206</td><td>Flangeless male nut Delrin, 1/4-28 flat-bottom, for 1/16&quot; OD blue</td></tr><tr><td>Function generator</td><td>Agilent Technologies</td><td>33120A</td><td>High-performance 15 MHz synthesized&amp;nbsp;function generator&amp;nbsp;with built-in arbitrary waveform capability</td></tr><tr><td>Goat anti-mouse IgG H&amp;amp;L (Alexa Fluor 488)</td><td>Abcam</td><td>ab150117</td><td>Goat anti-mouse IgG H&amp;amp;L (Alexa Fluor 488) preadsorbed</td></tr><tr><td>Goat anti-rabbit IgG H&amp;amp;L (Alexa Fluor 555)</td><td>Abcam</td><td>ab150086</td><td>Goat polyclonal secondary antibody to rabbit IgG - H&amp;amp;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&amp;nbsp;</td></tr><tr><td>Indium&amp;ndash;tin&amp;ndash;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&amp;nbsp;thermocouples</td></tr><tr><td>Luer adapter</td><td>IDEX Health &amp;amp; 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 &amp;nbsp;marker&amp;nbsp; O4&amp;nbsp; antibody</td><td>R&amp;amp;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&amp;nbsp;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&amp;ndash;integral&amp;ndash;derivative (PID) controller</td><td>Toho Electronics</td><td>TTM-J4-R-AB</td><td>Temperature controller&amp;nbsp;</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 &amp;amp; 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 suitable for confocal microscopy examinations. The MOE chip is also designed to investigate the effects of different cell culture conditions (e.g., multiple EF stimulation, several drugs, different coating substrate, multiple series of cells) simultaneously in a single experiment.
The mNPCs were exposed to square-wave DC pulses (magnitude 300 mV/mm at a frequency of 100 Hz). The DC pulse stimulation was conducted for 48 h. The differentiated cells were immunostained with Tuj1 (neuron-specific class III &#946;-tubulin), glial fibrillary acidic protein (GFAP to identify astrocytes), and oligodendrocyte marker O4. After the DC pulse treatment, the mNPCs expressed significantly high numbers of neurons (Tuj1+ cells) at DIV 7. At DIV 3, astrocytes (GFAP+ cells) were present at relatively higher levels in the stimulation groups than in the control (CTL) group. Compared with the CTL group, oligodendrocytes (O4+ cells) were significantly higher in the stimulation group at DIV 7 and DIV 14 (Figure 7). These results show that the DC pulse stimulation resulted in mNPCs differentiating into neurons, astrocytes, and oligodendrocytes simultaneously in stem cell maintenance medium. These results suggest that the MOE microfluidic system is suitable for a long-term cell differentiation study by microscopy.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61917/61917fig01.jpg" /> 
Figure 1: The detailed configuration of the multichannel optically transparent electrotactic chip. (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), and a cover glass (24 mm &#215; 60 mm). There are three cell culture chambers in the MOE chip. The MOE chip has connecting holes for the medium inlet/outlet and the agar salt bridges. Cells were cultured in the cell culture region (width 3 mm x length 42 mm x height 0.07 mm). Figure 1A has been modified from Chang et al.6. (B) Photograph of the MOE chip comprising adaptors, PMMA sheets, double-sided tape, and cover glass. Abbreviations: MOE= multichannel optically transparent electrotactic; PMMA = polymethyl methacrylate. <a href="https://www.jove.com/files/ftp_upload/61917/61917fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61917/61917fig02.jpg" /> 
Figure 2: The fabrication and assembling processes of the MOE chip. (A) The designed patterns of the PMMA sheets or double-sided tape were fabricated using laser micromachining. (B) The individual PMMA sheets were cut by a CO2 laser scriber. (C) The multiple layers of the cleaned PMMA sheets were bonded together by a thermal bonder. Abbreviations: MOE= multichannel optically transparent electrotactic; PMMA = polymethyl methacrylate; CO2 = carbon dioxide. <a href="https://www.jove.com/files/ftp_upload/61917/61917fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61917/61917fig03.jpg" /> 
Figure 3: A photograph of the MOE chip. This figure has been modified from Chang et al.6. Abbreviation: MOE= multichannel optically transparent electrotactic. <a href="https://www.jove.com/files/ftp_upload/61917/61917fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61917/61917fig04.jpg" /> 
Figure 4: Medium and electrical connection to the MOE chip. (A) Photograph of the components for the medium flow network and the EF network in the MOE microfluidic system, including the&#160;PTFE tube, flat-bottom connector, cone connector, cone-Luer adaptor, white finger-tight plug, Luer adaptor, Luer lock syringe, black rubber bung, and the Ag/AgCl electrodes. (B) Photograph of the configuration for the medium flow network. Abbreviations: MOE= multichannel optically transparent electrotactic; EF = electric field; PTFE = polytetrafluoroethylene; Ag = silver; AgCl = silver chloride. <a href="https://www.jove.com/files/ftp_upload/61917/61917fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/61917/61917fig05.jpg" /> 
Figure 5: A photograph showing the MOE chip on a microscope. Abbreviations: MOE= multichannel optically transparent electrotactic; Ag = silver; AgCl = silver chloride; ITO = indium-tin-oxide. <a href="https://www.jove.com/files/ftp_upload/61917/61917fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/61917/61917fig06.jpg" /> 
Figure 6: The configuration and the system used for the DC pulse stimulation. (A) The configuration of the entire system for the DC pulse stimulation. The syringes connected to the MOE chip were used for medium infusion and waste efflux. The DC pulse in the chip was provided by a power supply conducted through the Ag/AgCl electrodes. The device setup was installed on the X-Y-Z motorized stage of an inverted phase-contrast microscope equipped with a digital camera. (B) A photograph showing the setup on a laboratory bench. Abbreviations: MOE= multichannel optically transparent electrotactic; Ag = silver; AgCl = silver chloride; ITO = indium-tin-oxide; EF = electric field. <a href="https://www.jove.com/files/ftp_upload/61917/61917fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/61917/61917fig07.jpg" /> 
Figure 7: Differentiation of the mNPC cells in the control group (CTL) and in the DC pulse stimulation group at DIV 3, 7, and 14. The percentage of neuron (Tuj1+ cells), astrocytes (GFAP+ cells), and oligodendrocytes (O4+ cells) in (A-C) the CTL group and (D-F) in the stimulation (DC pulses) group. This figure has been published by Chang et al.1. Abbreviations: CTL: control; DC = direct current; Tuj1 = neuron-specific class III &#946;-tubulin; GFAP = glial fibrillary acidic protein; O4 = oligodendrocyte marker O4.&#160;<a href="https://www.jove.com/files/ftp_upload/61917/61917fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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

Universidad Científica del Sur

Research

JoVE Journal

Bioengineering

3.6K 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

Differentiation of a Human Neural Stem Cell Line on Three Dimensional Cultures, Analysis of MicroRNA and Putative Target Genes

Neural stem cells (NSCs) are capable of self-renewal and differentiation into neurons, astrocytes and oligodendrocytes under specific local microenvironments. In here, we present a set of methods used for three dimensional (3D) differentiation and miRNA analysis of a clonal human neural stem cell (hNSC) line, currently in clinical trials for stroke disability (NCT01151124 and NCT02117635, Clinicaltrials.gov). HNSCs were derived from an ethical approved first trimester human fetal cortex and conditionally immortalized using retroviral integration of a single copy of the c-mycERTAMconstruct. We describe how to measure axon process outgrowth of hNSCs differentiated on 3D scaffolds and how to quantify associated changes in miRNA expression using PCR array. Furthermore we exemplify computational analysis with the aim of selecting miRNA putative targets. SOX5 and NR4A3 were identified as suitable miRNA putative target of selected significantly down-regulated miRNAs in differentiated hNSC. MiRNA target validation was performed on SOX5 and NR4A3 3&#8217;UTRs by dual reporter plasmid transfection and dual luciferase assay.

With the intent of characterizing changes in miRNAs on differentiated human neural stem cells (hNSCs) we describe hNSC differentiation on a three dimensional system,the evaluation of changes in microRNA expression by miRNA PCR array, and computational analysis for miRNA target prediction and its validation by dual luciferase assay.

Neural stem cells (NSCs) are capable of self-renewal and differentiation into neurons, astrocytes and oligodendrocytes under specific local ...

Developmental Biology

Electric Field-controlled Directed Migration of Neural Progenitor Cells in 2D and 3D Environments

Endogenous electric fields (EFs) occur naturally in vivo and play a critical role during tissue/organ development and regeneration, including that of the central nervous system1,2. These endogenous EFs are generated by cellular regulation of ionic transport combined with the electrical resistance of cells and tissues. It has been reported that applied EF treatment can promote functional repair of spinal cord injuries in animals and humans3,4. In particular, EF-directed cell migration has been demonstrated in a wide variety of cell types5,6, including neural progenitor cells (NPCs)7,8. Application of direct current (DC) EFs is not a commonly available technique in most laboratories. We have described detailed protocols for the application of DC EFs to cell and tissue cultures previously5,11. Here we present a video demonstration of standard methods based on a calculated field strength to set up 2D and 3D environments for NPCs, and to investigate cellular responses to EF stimulation in both single cell growth conditions in 2D, and the organotypic spinal cord slice in 3D. The spinal cordslice is an ideal recipient tissue for studying NPC ex vivo behaviours, post-transplantation, because the cytoarchitectonic tissue organization is well preserved within these cultures9,10. Additionally, this ex vivo model also allows procedures that are not technically feasible to track cells in vivo using time-lapse recording at the single cell level. It is critically essential to evaluate cell behaviours in not only a 2D environment, but also in a 3D organotypic condition which mimicks the in vivo environment. This system will allow high-resolution imaging using cover glass-based dishes in tissue or organ culture with 3D tracking of single cell migration in vitro and ex vivo and can be an intermediate step before moving onto in vivo paradigms.

This protocol demonstrates methods used to establish 2D and 3D environments in custom-designed electrotactic chambers, which can track cells in vivo/ex vivo using time-lapse recording at the single cell level, in order to investigate galvanotaxis/electrotaxis and other cellular responses to direct current (DC) electric fields (EFs).

Endogenous electric fields (EFs) occur naturally in vivo and play a critical role during tissue/organ development and regeneration, including that of the ...

Assessing Neural Stem Cell Motility Using an Agarose Gel-based Microfluidic Device

While microfluidic technology is reaching a new level of maturity for macromolecular assays, cell-based assays are still at an infant stage1. This is largely due to the difficulty with which one can create a cell-compatible and steady microenvironment using conventional microfabrication techniques and materials. We address this problem via the introduction of a novel microfabrication material, agarose gel, as the base material for the microfluidic device. Agarose gel is highly malleable, and permeable to gas and nutrients necessary for cell survival, and thus an ideal material for cell-based assays. We have shown previously that agarose gel based devices have been successful in studying bacterial and neutrophil cell migration2. In this report, three parallel microfluidic channels are patterned in an agarose gel membrane of about 1mm thickness. Constant flows with media/buffer are maintained in the two side channels using a peristaltic pump. Cells are maintained in the center channel for observation. Since the nutrients and chemicals in the side channels are constantly diffusing from the side to center channel, the chemical environment of the center channel is easily controlled via the flow along the side channels. Using this device, we demonstrate that the movement of neural stem cells can be monitored optically with ease under various chemical conditions, and the experimental results show that the over expression of epidermal growth factor receptors (EGFR) enhances the motility of neural stem cells. Motility of neural stem cells is an important biomarker for assessing cells aggressiveness, thus tumorigenic factor3. Deciphering the mechanism underlying NSC motility will yield insight into both disorders of neural development and into brain cancer stem cell invasion.

We demonstrate that the over expression of epidermal growth factor receptors (EGFR) enhances the motility of neural stem cells(NSCs) using a novel agarose gel based microfluidic device. This technology can be readily adaptable to other mammalian cell systems where cell sources are scarce, such as human neural stem cells, and the turn around time is critical.

While microfluidic technology is reaching a new level of maturity for macromolecular assays, cell-based assays are still at an infant stage1. This is ...

Biology

A Galvanotaxis Assay for Analysis of Neural Precursor Cell Migration Kinetics in an Externally Applied Direct Current Electric Field

The discovery of neural stem and progenitor cells (collectively termed neural precursor cells) (NPCs) in the adult mammalian brain has led to a body of research aimed at utilizing the multipotent and proliferative properties of these cells for the development of neuroregenerative strategies. A critical step for the success of such strategies is the mobilization of NPCs toward a lesion site following exogenous transplantation or to enhance the response of the endogenous precursors that are found in the periventricular region of the CNS. Accordingly, it is essential to understand the mechanisms that promote, guide, and enhance NPC migration. Our work focuses on the utilization of direct current electric fields (dcEFs) to promote and direct NPC migration - a phenomenon known as galvanotaxis. Endogenous physiological electric fields function as critical cues for cell migration during normal development and wound repair. Pharmacological disruption of the trans-neural tube potential in axolotl embryos causes severe developmental malformations1. In the context of wound healing, the rate of repair of wounded cornea is directly correlated with the magnitude of the epithelial wound potential that arises after injury, as shown by pharmacological enhancement or disruption of this dcEF2-3. We have demonstrated that adult subependymal NPCs undergo rapid and directed cathodal migration in vitro when exposed to an externally applied dcEF. In this protocol we describe our lab's techniques for creating a simple and effective galvanotaxis assay for high-resolution, long-term observation of directed cell body translocation (migration) on a single-cell level. This assay would be suitable for investigating the mechanisms that regulate dcEF transduction into cellular motility through the use of transgenic or knockout mice, short interfering RNA, or specific receptor agonists/antagonists.

In this protocol we demonstrate how to construct custom chambers that permit the application of a direct current electric field to enable time-lapse imaging of adult brain derived neural precursor cell translocation during galvanotaxis.

The discovery of neural stem and progenitor  cells (collectively termed neural precursor cells) (NPCs) in the adult  mammalian brain has led to a body of ...

Neuroscience

Preparation of Neuronal Co-cultures with Single Cell Precision

Microfluidic embodiments of the Campenot chamber have attracted great interest from the neuroscience community. These interconnected co-culture platforms can be used to investigate a variety of questions, spanning developmental and functional neurobiology to infection and disease propagation. However, conventional systems require significant cellular inputs (many thousands per compartment), inadequate for studying low abundance cells, such as primary dopaminergic substantia nigra, spiral ganglia, and Drosophilia melanogaster neurons, and impractical for high throughput experimentation. The dense cultures are also highly locally entangled, with few outgrowths (&#60;10%) interconnecting the two cultures. In this paper straightforward microfluidic and patterning protocols are described which address these challenges: (i) a microfluidic single neuron arraying method, and (ii) a water masking method for plasma patterning biomaterial coatings to register neurons and promote outgrowth between compartments. Minimalistic neuronal co-cultures were prepared with high-level (&#62;85%) intercompartment connectivity and can be used for high throughput neurobiology experiments with single cell precision.

Protocols for single neuron microfluidic arraying and water masking for the in-chip plasma patterning of biomaterial coatings are described. Highly interconnected co-cultures can be prepared using minimal cell inputs.

Microfluidic embodiments of the Campenot chamber have attracted great interest from the neuroscience community. These interconnected co-culture platforms ...

Microfluidic Device for the Separation of Non-Metastatic (MCF-7) and Non-Tumor (MCF-10A) Breast Cancer Cells Using AC Dielectrophoresis

Dielectrophoretic devices are capable of the detection and manipulation of cancer cells in a label-free, cost-effective, robust, and accurate manner using the principle of the polarization of the cancer cells in the sample volume by applying an external electric field. This article demonstrates how a microfluidic platform can be utilized for high-throughput continuous sorting of non-metastatic breast cancer cells (MCF-7) and non-tumor breast epithelial cells (MCF-10A) using hydrodynamic dielectrophoresis (HDEP) from the cell mixture. By generating an electric field between two electrodes placed side-by-side with a micron-sized gap between them in an HDEP microfluidic chip, non-tumor breast epithelial cells (MCF-10A) can be pushed away, exhibiting negative DEP inside the main channel, while the non-metastatic breast cancer cells follow their course unaffected when suspended in cell medium due to having conductivity higher than the membrane conductivity. To demonstrate this concept, simulations were performed for different values of medium conductivity, and the sorting of cells was studied. A parametric study was carried out, and a suitable cell mixture conductivity was found to be 0.4 S/m. By keeping the medium conductivity fixed, an adequate AC frequency of 0.8 MHz was established, giving maximum sorting efficiency, by varying the electric field frequency. Using the demonstrated method, after choosing the appropriate cell mixture suspension medium conductivity and frequency of the applied AC, maximum sorting efficiency can be achieved.

Microfluidic Device for the Separation of Non-Metastatic MCF-7 and Non-Tumor MCF-10A Breast Cancer Cells Using AC Dielectrophoresis

Breast cancer cells exhibit different dielectric properties compared to non-tumor breast epithelial cells. It has been hypothesized that, based on this difference in dielectric properties, the two populations can be separated for immunotherapy purposes. To support this, we model a microfluidic device to sort MCF-7 and MCF-10A cells.

Dielectrophoretic devices are capable of the detection and manipulation of cancer cells in a label-free, cost-effective, robust, and accurate manner using ...

Engineering

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.

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

Neuronal Differentiation from Mouse Embryonic Stem Cells In vitro

The neural differentiation of mouse embryonic stem cells (mESCs) is a potential tool for elucidating the key mechanisms involved in neurogenesis and potentially aid in regenerative medicine. Here, we established an efficient and low cost method for neuronal differentiation from mESCs in vitro, using the strategy of combinatorial screening. Under the conditions defined here, the 2-day embryoid body formation + 6-day retinoic acid induction protocol permits fast and efficient differentiation from mESCs into neural precursor cells (NPCs), as seen by the formation of well-stacked and neurite-like A2lox and 129 derivatives that are Nestin positive. The healthy state of embryoid bodies and the timepoint at which retinoic acid (RA) is applied, as well as the RA concentrations, are critical in the process. In the subsequent differentiation from NPCs into neurons, N2B27 medium II (supplemented by Neurobasal medium) could better support the long term maintenance and maturation of neuronal cells. The presented method is highly efficiency, low cost and easy to operate, and can be a powerful tool for neurobiology and developmental biology research.

Here, we established a low cost and easy to operate method that directs fast and efficient differentiation from embryonic stem cells into neurons. This method is suitable for popularization among laboratories and can be a useful tool for neurological research.

The neural differentiation of mouse embryonic stem cells (mESCs) is a potential tool for elucidating the key mechanisms involved in neurogenesis and ...

Electrical Stimulation-Induced Differentiation of Neural Stem and Progenitor Cells

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.

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

	Draw patterns for individual polymethyl methacrylate ...

JoVE Encyclopedia of Experiments

Neuronal Culture Techniques

Advanced Neuronal Models

Generating Neuromuscular Junctions Using a Microfluidic Device

To plate the neural progenitor cells, or NPCs, in the microfluidic device, remove DPBS from two wells on one side of the microgrooves in the device using a 200-microliter pipette. To seed a total of 250,000 NPCs in 60 to 100 microliters of day 10 motor neuron medium per device in the top right well, seed half of the cell suspension close to the channel opening at an angle of 45 degrees.
Pause for a few seconds to allow the cell suspension to flow through the channel before adding the remaining half of the cell suspension in the lower well. Use a pen to mark the seeded side as NPC or equivalent for easy orientation of the device without a microscope and incubate for five minutes for cell attachment. Next, slowly top up the two NPC-seeded wells with an additional day 10 motor neuron medium to a total volume of 200 microliters per well. Using a 200-microliter pipette, remove DPBS from the two wells on the other side of the microgrooves opposite to the freshly seeded NPCs.
Add 200 microliters of day 10 motor neuron media per well to the unseeded wells. Then, add 6 milliliters of DPBS per 10-centimeter dish around the device to prevent evaporation of the medium during incubation.
To change the medium, slowly remove media in both wells with NPCs by positioning the 200-microliter pipette tip at the bottom edge of the well wall opposite the channel opening. To prevent a strong medium flow from damaging the cells in the channel, slowly, add 50 to 100 microliters of fresh motor neuron medium to each well, by continuously changing between the top and bottom well. Repeat the process until each well contains 200 microliters of medium.
On day 17 of the motor neuron differentiation, remove the motor neuron medium on the unseeded side of the microgrooves in the device with a 200-microliter pipette and wash the wells with DPBS. Seed a total of 200,000 human primary mesoangioblasts or MABs in 60 to 100 microliters of growth medium per device by seeding half of the cell suspension in the top well.
Pause for a few seconds to allow the cell suspension to flow through the channel before seeding the remaining half of the cell suspension in the bottom well, as demonstrated earlier for plating NPCs. Incubate the device for five minutes for cell attachment. Then, top up the two freshly MAB-seeded wells with an additional growth medium and incubate again as demonstrated.
To initiate the chemotactic and volumetric gradient on the 21st day of the motor neuron differentiation, add 200 microliters per well of motor neuron basal medium containing growth factors to the myotube compartment. Then, add 100 microliters per well of motor neuron basal medium without growth factors to the motor neuron compartment.