Name: electrotaxis studies of lung cancer cells using a multichannel dual-electric-field microfluidic chip
Uploaded: 2015-12-29T15:00:00
Description: Watch this Scientific Journal Video about Electrotaxis Studies of Lung Cancer Cells using a Multichannel Dual-electric-field Microfluidic Chip at JoVE.com

This work is financially supported by the Ministry of Science and Technology, Taiwan (Contract no. MOST 103-2113-M-001 -003 -MY2) and the Research Program on Nanoscience and Nanotechnology, Academia Sinica, Taiwan.

1. Design and Fabrication of the MDF Chip
<ol>
	<li>Draw an individual acrylic layer pattern using commercial software, such as AutoCAD, and save the pattern.
	<ol>
		<li>Review the design of the four-layer acrylic sheet pattern in Figure 1A and confirm the inter-layer connections.</li>
	</ol>
	</li>
	<li>Fabricate all the acrylic sheets and the double-sided tape by laser ablation using a CO2 laser scriber (Figures 1B, 2A, and 2B).16</s...

<ol>
	<li>McCaig, C. D., Rajnicek, A. M., Song, B., Zhao, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlling+cell+behavior+electrically:+current+views+and+future+potential.">Controlling cell behavior electrically: current views and future potential.</a> Physiol Rev. 85, 943-978 (2005).</li><li>Djamgoz, M. B. A., Mycielska, M., Madeja, Z., Fraser, S. 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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrotaxis+of+lung+cancer+cells+in+a+multiple-electric-field+chip.">Electrotaxis of lung cancer cells in a multiple-electric-field chip.</a> Biosens Bioelectron. 24, 3510-3516 (2009).</li><li>Huang, C. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+expression+of+human+lung+cancer+cell+line+CL1-5+in+response+to+a+direct+current+electric+field.">Gene expression of human lung cancer cell line CL1-5 in response to a direct current electric field.</a> PLoS One. 6, 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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrotaxis+of+lung+cancer+cells+in+ordered+three-dimensional+scaffolds.">Electrotaxis of lung cancer cells in ordered three-dimensional scaffolds.</a> Biomicrofluidics. 6, 14102-1410214 (2012).</li><li>Tsai, H. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+EGFR+and+RTK+signaling+in+the+electrotaxis+of+lung+adenocarcinoma+cells+under+direct-current+electric+field+stimulation.">Evaluation of EGFR and RTK signaling in the electrotaxis of lung adenocarcinoma cells under direct-current electric field stimulation.</a> PLoS One. 8, e73418 (2013).</li><li>Hou, H. S., Tsai, H. F., Chiu, H. T., Cheng, J. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+chemical+and+electrical+stimulation+on+lung+cancer+cells+using+a+multichannel-dual-electric-field+chip.">Simultaneous chemical and electrical stimulation on lung cancer cells using a multichannel-dual-electric-field chip.</a> Biomicrofluidics. 8, (2014).</li><li>Faupel, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electropotential+evaluation+as+a+new+technique+for+diagnosing+breast+lesions.">Electropotential evaluation as a new technique for diagnosing breast lesions.</a> Eur J Radiol. 24, 33-38 (1997).</li><li>Szatkowski, M., Mycielska, M., Knowles, R., Kho, A. L., Djamgoz, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophysiological+recordings+from+the+rat+prostate+gland+in+vitro:+identified+single-cell+and+transepithelial+(lumen)+potentials.">Electrophysiological recordings from the rat prostate gland in vitro: identified single-cell and transepithelial (lumen) potentials.</a> BJU Int. 86, 1068-1075 (2000).</li><li>McCaig, C. D., Song, B., Rajnicek, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrical+dimensions+in+cell+science.">Electrical dimensions in cell science.</a> J Cell Sci. 122, 4267-4276 (2009).</li><li>Das, T., Maiti, T. K., Chakraborty, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Traction+force+microscopy+on-chip:+shear+deformation+of+fibroblast+cells.">Traction force microscopy on-chip: shear deformation of fibroblast cells.</a> Lab Chip. 8, 1308-1318 (2008).</li><li>Lin, F., Butcher, E. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T+cell+chemotaxis+in+a+simple+microfluidic+device.">T cell chemotaxis in a simple microfluidic device.</a> Lab Chip. 6, 1462-1469 (2006).</li><li>Li, J., Zhu, L., Zhang, M., Lin, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+device+for+studying+cell+migration+in+single+or+co-existing+chemical+gradients+and+electric+fields.">Microfluidic device for studying cell migration in single or co-existing chemical gradients and electric fields.</a> Biomicrofluidics. 6, 24121-2412113 (2012).</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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrotaxis+of+oral+squamous+cell+carcinoma+cells+in+a+multiple-electric-field+chip+with+uniform+flow+field.">Electrotaxis of oral squamous cell carcinoma cells in a multiple-electric-field chip with uniform flow field.</a> Biomicrofluidics. 6, 34116 (2012).</li><li>Cheng, J. Y., Wei, C. W., Hsu, K. H., Young, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct-write+laser+micromachining+and+universal+surface+modification+of+PMMA+for+device+development.">Direct-write laser micromachining and universal surface modification of PMMA for device development.</a> Sensors and Actuators B: Chemical. 99, 186-196 (2004).</li><li>Chu, Y. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selection+of+invasive+and+metastatic+subpopulations+from+a+human+lung+adenocarcinoma+cell+line.">Selection of invasive and metastatic subpopulations from a human lung adenocarcinoma cell line.</a> Am J Respir Cell Mol Biol. 17, 353-360 (1997).</li><li>Cheng, J. Y., Yen, M. H., Kuo, C. T., Young, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+transparent+cell-culture+microchamber+with+a+variably+controlled+concentration+gradient+generator+and+flow+field+rectifier.">A transparent cell-culture microchamber with a variably controlled concentration gradient generator and flow field rectifier.</a> Biomicrofluidics. 2, 24105 (2008).</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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ITO+patterning+by+a+low+power+Q-switched+green+laser+and+its+use+in+the+fabrication+of+a+transparent+flow+meter.">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, 2316 (2007).</li><li>Pu, J., Zhao, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Golgi+polarization+in+a+strong+electric+field.">Golgi polarization in a strong electric field.</a> J Cell Sci. 118, 1117-1128 (2005).</li><li>Zhao, M., Bai, H., Wang, E., Forrester, J. V., McCaig, C. 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We found the process of adhering acrylic adaptors onto Layer 1 of the MDF chip to be tricky. The application of just 1 to 2 &#956;l of super glue is sufficient to firmly adhere the adaptor onto the MDF chip. Larger amounts of glue resulted in an incomplete polymerization of the super glue and failure to adhere. Once the acrylic adaptors were firmly adhered onto the MDF chip, liquid leakage in the microfluidic system rarely occurred. In addition, O/N&#160;incubation inside the vacuum chamber helped to remove the air trapp...

The behavior of adherent cells moving toward an anode or cathode under a direct current electric-field (dcEF) is referred to as electrotaxis. The electrotactic behavior of cells plays a significant role in embryogenesis, nerve regeneration, and wound healing.1 Tumor cells such as rat prostate cancer cells,2 breast cancer cells,3 and lung adenocarcinoma cells4-8 have shown electrotactic movement under an applied dcEF. The physiological EF has been measured in gland tissues.9,10 Electrotaxis has also been reported in gland-associated tumor cells.2,3 Taken together, the electrotaxis of cancer cells is considered to be a metastasis factor.11 Controlling the electrical guidance of cancer cells under dcEF may be a potential approach for the future treatment of cancer. However, today, the detailed molecular mechanism of electrotaxis remains controversial. Therefore, an investigation of the influence of electrical stimulation on cancer cell migration can facilitate the development of strategies for cancer treatment.
Recently, bio-microfluidic devices have been fabricated for studying cellular responses to flow shear force,12 chemical gradients,13 and electrical stimuli4 in vitro. The fabrication of bio-microfluidic devices using polydimethylsiloxane (PDMS) or polymethylmethacrylate (PMMA, also known as acrylic) has successfully reduced the failure rate of such experiments. Moreover, using acrylic-based microfluidic devices as a prototype for investigating biological subjects is simpler than using PDMS chips. Various functions in acrylic-based devices have been developed for electrotaxis study. However, none of the previous designs are able to simultaneously test the effects of various chemical conditions and the electric-field on cells for electrotaxis study. Thus, we developed a microfluidic device-the multichannel dual-electric-field (MDF) chip-containing four independent culture channels and eight different experimental conditions in one chip.
The acrylic-based MDF chip, first reported by Hou et al.,8 integrates electrical stimulation and several chemically isolated channels. These chemically isolated channels can be used to culture different types of cells in one experiment. The dcEF in the channels is produced by an electrical power supply. Two independent electric fields, one with applied electric-field strength (EFS) and another with 0 EFS, are conducted in each chemically isolated channel. In this way, the chip provides better-controlled coexisting EF and chemical stimulation. Furthermore, results from the numerical simulation of the chemical diffusion inside the MDF chip indicate that no cross contamination occurred between the channels after a 24 hr experimental period.8
Compared to the device reported by Li et al.,14 the MDF chip provides a larger culture area, which allows for further biochemical analysis of the electrically stimulated cells. Additionally, with the MDF chip&#39;s larger observation area, more cells can be observed in the test, so the analysis of migration speed or directedness of the electrically stimulated cells is more accurate. The single-channel chip designs of previous studies reported by Huang et al.4 and Tsai et al.15 allow only one type of cell or chemical to be tested. However, the MDF chip can be used to investigate the effects of various chemicals on electrotaxis, as well as the effects of electrical stimulation on different types of cells. In other words, the MDF chip allows for the efficient study of chemical dose dependencies.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr >
			<td >Reagent</td>
			<td ></td>
			<td ></td>
			<td ></td>
		</tr>
		<tr >
			<td >DMEM medium</td>
			<td >Gibco,Invitrogen, USA</td>
			<td >12800-017</td>
			<td></td>
		</tr>
		<tr >
			<td >Fetal Bovine Serum</td>
			<td >Gibco,Invitrogen, USA</td>
			<td >16000-044</td>
			<td></td>
		</tr>
		<tr >
			<td >Trypsin</td>
			<td >Gibco,Invitrogen, USA</td>
			<td >25200-072</td>
			<td></td>
		</tr>
		<tr >
			<td >PBS</td>
			<td >Basic Life</td>
			<td >BL2651</td>
			<td></td>
		</tr>
		<tr >
			<td >Y-27632 (hydrochloride)</td>
			<td >Cayman Chemical Co</td>
			<td >10005583</td>
			<td></td>
		</tr>
		<tr >
			<td >agarose</td>
			<td >LONZO, USA</td>
			<td >SeaKem LE AGAROSE</td>
			<td></td>
		</tr>
		<tr >
			<td >syringe</td>
			<td>Terumo</td>
			<td>3 ml with Luer taper</td>
			<td></td>
		</tr>
		<tr >
			<td >3-way stopcock</td>
			<td>Nipro</td>
			<td >with Luer taper</td>
			<td></td>
		</tr>
		<tr >
			<td >PMMA (acrylic)</td>
			<td >HiShiRon Industries CO., Ltd, Taiwan</td>
			<td ></td>
			<td >thickness 1mm, 2mm</td>
		</tr>
		<tr >
			<td >acrylic adaptor</td>
			<td >KuanMin Technology Co., Ltd, Taichung, Taiwan</td>
			<td >1/4-28 port, 10x10x6 mm</td>
			<td>customized</td>
		</tr>
		<tr >
			<td >nut</td>
			<td >Thermo Fisher Scientific Inc.</td>
			<td >UPCHURCH:P-206x, P-200x, F120x, P-659, P-315x</td>
			<td></td>
		</tr>
		<tr >
			<td >Microscope cover glass</td>
			<td >Deckgl&#228;ser, Germany</td>
			<td >24x60 mm</td>
			<td></td>
		</tr>
		<tr >
			<td >double-sided tape</td>
			<td >3M</td>
			<td >PET 8018</td>
			<td></td>
		</tr>
		<tr >
			<td >super glue</td>
			<td >3M</td>
			<td >Scotch Liquid Plus Super Glue</td>
			<td></td>
		</tr>
		<tr >
			<td >Teflon tube</td>
			<td >HENG YI ENTERPRISE CO., LTD., Taiwan</td>
			<td >UPTB_06, DUPONT TEFLON BRAND RESIN FEP TUBING</td>
			<td>outer diameter 1/16 in., inner diameter 0.03 in.; Upchurch Scienti&#64257;c</td>
		</tr>
		<tr >
			<td >TFD4 detergent</td>
			<td >Franklab, France</td>
			<td >TFD4</td>
			<td></td>
		</tr>
		<tr >
			<td >ultrasonic steri cleaner</td>
			<td >LEO ULTRASONIC CO., LTD., Taiwan</td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Thermo bonder</td>
			<td >KuanMin Technology Co., Ltd, Taichung, Taiwan</td>
			<td>customized</td>
			<td></td>
		</tr>
		<tr >
			<td >CO2 laser scriber</td>
			<td >LTT group, Taiwan</td>
			<td >ISL-II</td>
			<td></td>
		</tr>
		<tr >
			<td >indium tin oxide glass (ITO glass)</td>
			<td >AimCore Technology Co., Ltd</td>
			<td >TN/STN, &#8806;10&#937;</td>
			<td></td>
		</tr>
		<tr >
			<td >proportional-integral-derivative (PID) controller</td>
			<td>JETEC Electronics Co., Japen</td>
			<td >TTM-J40-R-AB,</td>
			<td></td>
		</tr>
		<tr >
			<td >K-type thermocouple</td>
			<td >TECPEL</td>
			<td >TPK-02A</td>
			<td></td>
		</tr>
		<tr >
			<td >4-channel syringe pump</td>
			<td >KdScientific, USA</td>
			<td >250P</td>
			<td></td>
		</tr>
		<tr >
			<td >DC power supply</td>
			<td >GWInstek, Taiwan</td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >X-Y-Z motor stage</td>
			<td >TanLian, E-O Co. Ltd., Taiwan</td>
			<td>customized</td>
			<td></td>
		</tr>
		<tr >
			<td >inverted microscope</td>
			<td >Olympus, Japan</td>
			<td >CKX41</td>
			<td></td>
		</tr>
		<tr >
			<td >digital SLR camera</td>
			<td >Canon, Japan</td>
			<td >60D</td>
			<td></td>
		</tr>
	</tbody>
</table>

electrotaxis studies of lung cancer cells using a multichannel dual-electric-field microfluidic chip

The behavior of directional cell migration under a direct current electric-field (dcEF) is referred to as electrotaxis. The significant role of physiological dcEF in guiding cell movement during embryo development, cell differentiation, and wound healing has been demonstrated in many studies. By applying microfluidic chips to an electrotaxis assay, the investigation process is shortened and experimental errors are minimized. In recent years, microfluidic devices made of polymeric substances (e.g., polymethylmethacrylate, PMMA, or acrylic) or polydimethylsiloxane (PDMS) have been widely used in studying the responses of cells to electrical stimulation. However, unlike the numerous steps required to fabricate a PDMS device, the simple and rapid construction of the acrylic micro&#64258;uidic chip makes it suitable for both device prototyping and production. Yet none of the reported devices facilitate the efficient study of the simultaneous chemical and dcEF effects on cells. In this report, we describe our design and fabrication of an acrylic-based multichannel dual-electric-field (MDF) chip to investigate the concurrent effect of chemical and electrical stimulation on lung cancer cells. The MDF chip provides eight combinations of electrical/chemical stimulations in a single test. The chip not only greatly shortens the required experimental time but also increases accuracy in electrotaxis studies.

Fabrication and assembly of the MDF device
A schematic diagram of the acrylic-based MDF chip is shown in Figure 1A. Four acrylic sheets, one cover glass, 13 acrylic adaptors, and a piece of double-sided tape were used in the assembly of the completed MDF chip (Figure 2D). There are only four independent culture channels in the MDF device. However, the on-chip salt bridge network...

Watch this Scientific Journal Video about Electrotaxis Studies of Lung Cancer Cells using a Multichannel Dual-electric-field Microfluidic Chip at JoVE.com

Electrotaxis Studies of Lung Cancer Cells using a Multichannel Dual-electric-field Microfluidic Chip

Many microfluidic devices have been developed for use in the study of electrotaxis. Yet, none of these chips allows the efficient study of the simultaneous chemical and electric-field (EF) effects on cells. We developed a polymethylmethacrylate-based device that offers better-controlled coexisting EF and chemical stimulation for use in electrotaxis research.

The behavior of directional cell migration under a direct current electric-field (dcEF) is referred to as electrotaxis. The significant role of ...

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