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
	<ol>
		<li>Turn on the laser scriber and connect the machine to the controlling laptop.</li>
		<li>Run the commercial software and open the pattern designed in step 1.1 by clicking &#34;designed pattern file.&#34;</li>
		<li>Place a piece of blank acrylic sheet or double-sided tape on the X-Y-Z stage of the laser scriber.</li>
		<li>Set the focus of the laser beam on the surface of the acrylic sheet or double-sided tape with an auto-alignment stick provided by the manufacturer of the laser scriber.</li>
		<li>Send the designed pattern to the laser scriber for direct machining of the acrylic sheet or double-sided tape.</li>
	</ol>
	</li>
	<li>Remove the protective paper from the acrylic sheets using forceps and blow the surface clean with nitrogen gas.</li>
	<li>Stack the acrylic sheets and bond them together under a pressure of 2&#160;kg/cm2 in a thermal bonder for 45 min at 110 &#176;C to form the flow/electrical stimulation channel assembly.</li>
	<li>Prepare the microscope cover glass as the cell culture substrate in the chip.
	<ol>
		<li>Put the cover glass into a staining jar and fill the jar with a ten-fold dilution of the detergent listed in the material list.</li>
		<li>Put the cover glass and staining jar in an ultrasonic steri-cleaner and clean the cover glass for 15 min.</li>
		<li>Pour the diluted detergent out of the staining jar, refill the jar with distilled water, and repeat step 1.5.2 3 times.</li>
		<li>Dry the cleaned cover glass by blowing it with nitrogen gas before adhering it to the double-sided tape.</li>
	</ol>
	</li>
	<li>Adhere the cleaned cover glass to the flow/electrical stimulation channel assembly with the double-sided tape patterned in step 1.2.</li>
	<li>Adhere 13 pieces of acrylic adaptors to the individual openings in Layer 1 of the MDF chip assembly with super glue. The MDF chip assembly is then complete (Figure 2D).</li>
	<li>Sterilize the complete MDF chip assembly with 30 min of UV irradiation prior to use.</li>
</ol>
2. Setup of the Salt Bridge Network of the MDF Chip
<ol>
	<li>Prior to use, sterilize all the plastic tubes, finger-tight nuts, and microcentrifuge tubes shown in Figure 3B by setting a holding time of 15 min at 121 &#176;C in the autoclave.</li>
	<li>Connect the fluoroplastic tubes (Figure 3B-i) to the MDF chip assembly via the medium inlet and outlet adaptors shown in Figure 1A.</li>
	<li>Connect the Luer taper of the medium inlet and outlet plastic tubes in Figure 3B-i to the 3-way stopcocks.</li>
	<li>Take 2.5 ml of CO2-equilibrated phosphate-buffered saline (PBS) using a 3 ml syringe and connect the syringe to the 3-way stopcock of the inlet plastic tube. Connect an empty 3 ml syringe to the 3-way stopcock of the outlet plastic tube. The two syringes are thus interconnected through the MDF chip. 
	NOTE: Incubate the PBS in the 37 &#176;C 5%-CO2 cell culture incubator O/N to obtain CO2-equilibrated PBS.</li>
	<li>Seal the openings of the blue and the green adaptors (Figure 1A) on the Layer 1 PMMA sheet with white solid finger-tight nuts (Figure 3B-iii).</li>
	<li>Fill the salt bridge channels and culture chambers with the CO2-equilibrated PBS in the syringe described in setup step 2.4. Avoid the formation of bubbles.</li>
	<li>Next, put the CO2-equilibrated PBS-containing MDF chip in the 37 &#176;C 5%-CO2 cell culture incubator for O/N incubation. This allows the dissolved air in the double-sided tape to form bubbles in the chambers.</li>
	<li>Flush away the bubbles in the channels by a rapid PBS flow in the channel using the two syringes. Pump the PBS back and forth if necessary.</li>
	<li>Drain the PBS in the channels via the 3-way stopcock connected to the medium outlet tube.</li>
	<li>Using a new 3-ml syringe, take 2.5 ml of CO2-equilibrated Dulbecco&#39;s Modified Eagle Medium (DMEM) (see step 3.5), and replace the syringe connected to the inlet with the new one. Refill the channels with the medium.</li>
	<li>Preparation of the salt bridge network.
	<ol>
		<li>Temporally remove the solid nuts (Figure 3B-iii) on the green adaptors (Figure 1A) and inject 3% hot (&#62;70 &#176;C) agarose into the salt bridge channel through the opening in the green adaptors (Figure 1A). 
		NOTE: Preparation of hot agarose: Dissolve 1.5 g of agarose powder in 50 ml of PBS. Sterilize by setting a holding time of 20 min at 121 &#176;C in the autoclave.</li>
		<li>Stop injecting the agarose when the liquid fills three-quarters of the length of the salt bridge channel.</li>
		<li>Seal the pores on the green adaptors (Figure 1A) by screwing the solid nuts (Figure 3B-iii) after the agarose injection.</li>
		<li>Replace the solid nuts on the blue adaptors (Figure 1A) with the translucent tubular finger-tight nuts (Figure 3B).</li>
		<li>Load the 3% pre-heated agarose into the tubular nuts (Figure 3B-iv).</li>
		<li>Embed the Ag/AgCl electrodes (Figure 3B-v) into the tubular nuts (Figure 3B-iv) before the solidification of the agarose.</li>
	</ol>
	</li>
	<li>After completing the setup of the salt bridge network, inject the CL1-5 lung cancer cells into the culture chambers, one chamber at a time.</li>
</ol>
3. Preparation of Cancer Cells and Set Up for Electrotactic Experiment
<ol>
	<li>Regular culture of lung cancer cell line CL1-5.
	<ol>
		<li>Culture the CL1-5 lung cancer cells, obtained from Prof. Pan-Chyr Yang,17 in the complete medium in a 75T cell culture flask at 37 &#176;C in a 5% CO2 atmosphere. The complete medium is composed of DMEM and 10% fetal bovine serum (FBS). Sub-culture the cells every 3 to 4 days. The cells used for performing electrotaxis experiments are less than 25 passages from the original source.</li>
	</ol>
	</li>
	<li>Add 2 ml of 0.25% trypsin buffer to the exponentially growing CL1-5 cells and incubate for 2 min in the 37 &#176;C 5%-CO2 cell culture incubator for cell detachment.</li>
	<li>Terminate the detachment process with 6 ml of 10% FBS DMEM and centrifuge the cells at 300 g for 5 min at RT. Then discard the medium and suspend the cell pellet with 5 ml of PBS.</li>
	<li>Count the number of cells in the PBS. Then take 1 x 106 cells and centrifuge at 300 g for 5 min at RT.</li>
	<li>Discard the PBS. Suspend the cells with pre-warmed CO2-equilibrated DMEM and adjust the cell density to be 1 &#215; 106 cells/ml. 
	NOTE: Incubate the complete medium in the 37 &#176;C 5%-CO2 cell culture incubator O/N to obtain the CO2-equilibrated DMEM.</li>
</ol>
4. Set Up for Electrotactic Experiment
<ol>
	<li>From the outlet of the MDF chip, inject 0.3 ml, 1 &#215; 106/ml, of the cells into the chip.</li>
	<li>Incubate the cell-seeded MDF chip in the cell culture incubator (set at 37 &#176;C and 5% CO2) for 2-4 hr.</li>
	<li>Setup of MDF microfluidic system.
	<ol>
		<li>Install the MDF microfluidic system onto a transparent indium tin oxide glass (ITO) heater. Measure the temperature of the MDF chip with a K-type thermocouple clipped between the MDF chip and the ITO glass. Control the ITO heater with a proportional-integral-derivative (PID) controller. Set the MDF microfluidic system incubation temperature to 37 &#176;C with the PID controller. 
		NOTE: The fabrication of the ITO heater and the setup of the cell culture heating system are described in previous reports.18,19</li>
		<li>Mount the temperature-controlled MDF chip assembly on the computer-controlled X-Y-Z motorized stage on an inverted microscope. The usage of the motorized stage is described in step 5.1. Pump the complete medium into the culture chambers through the inlets with a four-channel syringe pump.</li>
	</ol>
	</li>
	<li>Pump the complete medium into the MDF chip at a flow rate of 20 &#956;l/hr. The culture waste from the outlets is collected in the microcentrifuge tubes (shown as &#34;waste&#34; in Figure 3A).</li>
	<li>Incubate the cells for another 16-18 hr at 37 &#176;C in the MDF chip.</li>
	<li>To replace the medium, pump the medium containing 50, 25, 5, and 0 &#181;M kinase inhibitor, respectively, into each culture chamber at a flow rate of 20 &#956;l/min for 10 min. 
	NOTE: Prepare a 10 mM stock solution of the Rho-associated protein kinase inhibitor Y27632 by adding 3 ml of sterile water to the 10.6 mg kinase inhibitor. Dilute the 10 mM stock solution to 50, 25, and 5 &#181;M, respectively, using 10% FBS DMEM medium.</li>
	<li>Incubate the cells in the kinase inhibitor for another 60 min at 37 &#176;C. Set the medium flow rate to 20 &#956;l/hr. Use the same culture temperature and flow rate described above in later steps.</li>
	<li>Use electrical wires to connect a DC power supply to the MDF chip via the Ag/AgCl electrodes on the chip.</li>
	<li>Serially connect an ammeter in the electric circuit. Use the ammeter to monitor the electric current in the MDF chip.</li>
	<li>Turn on the DC power supply and set the ammeter current to 86.94 &#956;A by adjusting the power supply voltage to between 15 and 19 V. 
	NOTE: Considering Ohm&#39;s law, E = I/(&#963;Aeff), where I is the electric current flowing through the bulk material, i.e., the culture medium in the electrotactic chamber, &#963; (= 1.38 &#937;&#8722;1m&#8722;1) is the conductivity of the culture medium, Aeff (= 0.21 mm2 for a 3 mm width &#215; 0.07 mm height) is the effective cross-sectional area of the electrotactic chamber, and the electric current (I) required to generate a 300 mV/mm EF in the MDF chip is 86.94 &#956;A.</li>
</ol>
5. Acquisition and Analysis of Cell Images
<ol>
	<li>Control the motorized stage using a MATLAB GUI program. With the MDF microfluidic system mounted on the microscope, move the chip to the observation regions using the motorized stage.</li>
	<li>Record cell images using a digital single-lens reflex (SLR) camera mounted on the microscope.</li>
	<li>Take microscopic images using a 4X objective lens at intervals of 15 min for 2 hr.</li>
	<li>Cell migration analysis.
	<ol>
		<li>Run NIH ImageJ 1.47 V.</li>
		<li>Analyze the distance from the initial to the final positions of the cell&#39;s centroid.</li>
		<li>Go to &#34;File&#34;&#8594;&#34;Import&#34;&#8594;&#34;Image Sequence,&#34; and import nine images for cell migration analysis. The first image is the result of zero time, and the last image is the result of 120 min.</li>
		<li>Go to &#34;Analyze&#34;&#8594;&#34;Set Measurements&#34; and check the box next to &#34;Centroid&#34;</li>
		<li>Click on the &#34;Freehand Selections&#34; icon.</li>
		<li>Depict the edge of the selected cells in the first image.</li>
		<li>Go to &#34;Edit&#34;&#8594;&#34;Selection&#34;&#8594;&#34;Add to Manager&#34;</li>
		<li>Go to the ninth image and depict the edge of the same cells selected on the first image. The cell positions can be traced using the second to eighth images.</li>
		<li>Go to &#34;Edit&#34;&#8594;&#34;Selection&#34;&#8594;&#34;Add to Manager&#34;</li>
		<li>Repeat steps 5.4.5 to 5.4.9 to collect data from 90 to 100 cells.</li>
		<li>Go to &#34;Analyze&#34;&#8594;&#34;Measure&#34; to obtain the result of the initial and final positions of the selected cells. 
		NOTE: The migration speed is defined as the average cell movement length per hr. The directedness is defined as cosine, where is the angle between the vector of the dcEF (from anode to cathode) and the vector from the starting point of a cell to its final position. The directedness is &#8722;1 for cells migrating toward the anode and +1 for cells migrating toward the cathode. For a group of randomly migrating cells, the directedness is 0.2</li>
	</ol>
	</li>
</ol>

<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. P., Korohoda, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directional+movement+of+rat+prostate+cancer+cells+in+direct-current+electric+field:+involvement+of+voltagegated+Na++channel+activity.">Directional movement of rat prostate cancer cells in direct-current electric field: involvement of voltagegated Na+ channel activity.</a> J Cell Sci. 114, 2697-2705 (2001).</li><li>Pu, J., 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=EGF+receptor+signaling+is+essential+for+electric-field-directed+migration+of+breast+cancer+cells.">EGF receptor signaling is essential for electric-field-directed migration of breast cancer cells.</a> J Cell Sci. 120, 3395-3403 (2007).</li><li>Huang, C. W., Cheng, J. Y., Yen, M. 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=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. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrical+stimulation+directly+induces+pre-angiogenic+responses+in+vascular+endothelial+cells+by+signaling+through+VEGF+receptors.">Electrical stimulation directly induces pre-angiogenic responses in vascular endothelial cells by signaling through VEGF receptors.</a> J Cell Sci. 117, 397-405 (2004).</li><li>Yao, L., Shanley, L., McCaig, C., 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=Small+applied+electric+fields+guide+migration+of+hippocampal+neurons.">Small applied electric fields guide migration of hippocampal neurons.</a> J Cell Physiol. 216, 527-535 (2008).</li></ol>

The authors declare that they have no competing financial interests.

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 ...

electrotaxis-studies-lung-cancer-cells-using-multichannel-dual

Research

JoVE Journal

Bioengineering

8.6K Görüntüleme.  Academia Sinica. 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.

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

Fluorescence detection methods for microfluidic droplet platforms

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that accompany system miniaturisation. Advantages include the ability to efficiently process nano- to femoto- liter volumes of sample, facile integration of functional components, an intrinsic predisposition towards large-scale multiplexing, enhanced analytical throughput, improved control and reduced instrumental footprints.1
In recent years much interest has focussed on the development of droplet-based (or segmented flow) microfluidic systems and their potential as platforms in high-throughput experimentation.2-4 Here water-in-oil emulsions are made to spontaneously form in microfluidic channels as a result of capillary instabilities between the two immiscible phases. Importantly, microdroplets of precisely defined volumes and compositions can be generated at frequencies of several kHz. Furthermore, by encapsulating reagents of interest within isolated compartments separated by a continuous immiscible phase, both sample cross-talk and dispersion (diffusion- and Taylor-based) can be eliminated, which leads to minimal cross-contamination and the ability to time analytical processes with great accuracy. Additionally, since there is no contact between the contents of the droplets and the channel walls (which are wetted by the continuous phase) absorption and loss of reagents on the channel walls is prevented.
Once droplets of this kind have been generated and processed, it is necessary to extract the required analytical information. In this respect the detection method of choice should be rapid, provide high-sensitivity and low limits of detection, be applicable to a range of molecular species, be non-destructive and be able to be integrated with microfluidic devices in a facile manner. To address this need we have developed a suite of experimental tools and protocols that enable the extraction of large amounts of photophysical information from small-volume environments, and are applicable to the analysis of a wide range of physical, chemical and biological parameters. Herein two examples of these methods are presented and applied to the detection of single cells and the mapping of mixing processes inside picoliter-volume droplets. We report the entire experimental process including microfluidic chip fabrication, the optical setup and the process of droplet generation and detection.

Droplet-based microfluidic platforms are promising candidates for high throughput experimentation since they are able to generate picoliter, self-compartmentalized vessels inexpensively at kHz rates. Through integration with fast, sensitive and high resolution fluorescence spectroscopic methods, the large amounts of information generated within these systems can be efficiently extracted, harnessed and utilized.

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that ...

Microfluidic-based Electrotaxis for On-demand Quantitative Analysis of Caenorhabditis elegans' Locomotion

The nematode Caenorhabditis elegans is a versatile model organism for biomedical research because of its conservation of disease-related genes and pathways as well as its ease of cultivation. Several C. elegans disease models have been reported, including neurodegenerative disorders such as Parkinson's disease (PD), which involves the degeneration of dopaminergic (DA) neurons 1. Both transgenes and neurotoxic chemicals have been used to induce DA neurodegeneration and consequent movement defects in worms, allowing for investigations into the basis of neurodegeneration and screens for neuroprotective genes and compounds 2,3.
Screens in lower eukaryotes like C. elegans provide an efficient and economical means to identify compounds and genes affecting neuronal signaling. Conventional screens are typically performed manually and scored by visual inspection; consequently, they are time-consuming and prone to human errors. Additionally, most focus on cellular level analysis while ignoring locomotion, which is an especially important parameter for movement disorders.
We have developed a novel microfluidic screening system (Figure 1) that controls and quantifies C. elegans' locomotion using electric field stimuli inside microchannels. We have shown that a Direct Current (DC) field can robustly induce on-demand locomotion towards the cathode ("electrotaxis") 4. Reversing the field's polarity causes the worm to quickly reverse its direction as well. We have also shown that defects in dopaminergic and other sensory neurons alter the swimming response 5. Therefore, abnormalities in neuronal signaling can be determined using locomotion as a read-out. The movement response can be accurately quantified using a range of parameters such as swimming speed, body bending frequency and reversal time.
Our work has revealed that the electrotactic response varies with age. Specifically, young adults respond to a lower range of electric fields and move faster compared to larvae 4. These findings led us to design a new microfluidic device to passively sort worms by age and phenotype 6.
We have also tested the response of worms to pulsed DC and Alternating Current (AC) electric fields. Pulsed DC fields of various duty cycles effectively generated electrotaxis in both C. elegans and its cousin C. briggsae 7. In another experiment, symmetrical AC fields with frequencies ranging from 1 Hz to 3 KHz immobilized worms inside the channel 8.
Implementation of the electric field in a microfluidic environment enables rapid and automated execution of the electrotaxis assay. This approach promises to facilitate high-throughput genetic and chemical screens for factors affecting neuronal function and viability.

Microfluidic-based Electrotaxis for On-demand Quantitative Analysis of Caenorhabditis elegans&#039; Locomotion

A semi-automated micro-electro-fluidic method to induce on-demand locomotion in Caenorhabditis elegans is described. This method is based on the neurophysiologic phenomenon of worms responding to mild electric fields (&#x201C;electrotaxis&#x201D;) inside microfluidic channels. Microfluidic electrotaxis serves as a rapid, sensitive, low-cost, and scalable technique to screen for factors affecting neuronal health.

The nematode Caenorhabditis elegans is a versatile model organism for biomedical research because of its conservation of disease-related genes and ...

Microfluidic Chip Fabrication and Method to Detect Influenza

Fast and effective diagnostics play an important role in controlling infectious disease by enabling effective patient management and treatment. Here, we present an integrated microfluidic thermoplastic chip with the ability to amplify influenza A virus in patient nasopharyngeal (NP) swabs and aspirates. Upon loading the patient sample, the microfluidic device sequentially carries out on-chip cell lysis, RNA purification and concentration steps within the solid phase extraction (SPE), reverse transcription (RT) and polymerase chain reaction (PCR) in RT-PCR chambers, respectively. End-point detection is performed using an off-chip Bioanalyzer (Agilent Technologies, Santa Clara, CA). For peripherals, we used a single syringe pump to drive reagent and samples, while two thin film heaters were used as the heat sources for the RT and PCR chambers. The chip is designed to be single layer and suitable for high throughput manufacturing to reduce the fabrication time and cost. The microfluidic chip provides a platform to analyze a wide variety of virus and bacteria, limited only by changes in reagent design needed to detect new pathogens of interest.

An integrated microfluidic thermoplastic chip has been developed for use as a molecular diagnostic. The chip performs nucleic acid extraction, reverse transcriptase, and PCR. Methods for fabricating and running the chip are described.

Fast  and effective diagnostics play an important role in controlling infectious  disease by enabling effective patient management and treatment. Here, we ...

A Microfluidic Chip for the Versatile Chemical Analysis of Single Cells

We present a microfluidic device that enables the quantitative determination of intracellular biomolecules in multiple single cells in parallel. For this purpose, the cells are passively trapped in the middle of a microchamber. Upon activation of the control layer, the cell is isolated from the surrounding volume in a small chamber. The surrounding volume can then be exchanged without affecting the isolated cell. However, upon short opening and closing of the chamber, the solution in the chamber can be replaced within a few hundred milliseconds. Due to the reversibility of the chambers, the cells can be exposed to different solutions sequentially in a highly controllable fashion, e.g. for incubation, washing, and finally, cell lysis. The tightly sealed microchambers enable the retention of the lysate, minimize and control the dilution after cell lysis. Since lysis and analysis occur at the same location, high sensitivity is retained because no further dilution or loss of the analytes occurs during transport. The microchamber design therefore enables the reliable and reproducible analysis of very small copy numbers of intracellular molecules (attomoles, zeptomoles) released from individual cells. Furthermore, many microchambers can be arranged in an array format, allowing the analysis of many cells at once, given that suitable optical instruments are used for monitoring. We have already used the platform for proof-of-concept studies to analyze intracellular proteins, enzymes, cofactors and second messengers in either relative or absolute quantifiable manner.

In this article we present a microfluidic chip for single cell analysis. It allows the quantification of intracellular proteins, enzymes, cofactors, and second messengers by means of fluorescent assays or immunoassays.&#160;

We present a microfluidic device that enables the quantitative determination of intracellular biomolecules in multiple single cells in parallel. For this ...

A Microfluidic Chip for ICPMS Sample Introduction

This protocol discusses the fabrication and usage of a disposable low cost microfluidic chip as sample introduction system for inductively coupled plasma mass spectrometry (ICPMS). The chip produces monodisperse aqueous sample droplets in perfluorohexane (PFH). Size and frequency of the aqueous droplets can be varied in the range of 40 to 60 &#181;m and from 90 to 7,000&#160;Hz, respectively. The droplets are ejected from the chip with a second flow of PFH and remain intact during the ejection. A custom-built desolvation system removes the PFH and transports the droplets into the ICPMS. Here, very stable signals with a narrow intensity distribution can be measured, showing the monodispersity of the droplets. We show that the introduction system can be used to quantitatively determine iron in single bovine red blood cells. In the future, the capabilities of the introduction device can easily be extended by the integration of additional microfluidic modules.

We present a discrete droplet sample introduction system for inductively coupled plasma mass spectrometry (ICPMS). It is based on a cheap and disposable microfluidic chip that generates highly monodisperse droplets in a size range of 40&#8722;60 &#181;m at frequencies from 90 to 7,000 Hz.

This protocol discusses the fabrication and usage of a disposable low cost microfluidic chip as sample introduction system for inductively coupled plasma ...

The Multi-organ Chip - A Microfluidic Platform for Long-term Multi-tissue Coculture

The ever growing amount of new substances released onto the market and the limited predictability of current in vitro test systems has led to a high need for new solutions for substance testing. Many drugs that have been removed from the market due to drug-induced liver injury released their toxic potential only after several doses of chronic testing in humans. However, a controlled microenvironment is pivotal for long-term multiple dosing experiments,&#160;as even minor alterations in extracellular conditions may greatly influence the cell physiology. We focused within our research program on the generation of a microengineered bioreactor, which can be dynamically perfused by an on-chip pump and combines at least two culture spaces for multi-organ applications. This circulatory system mimics the in vivo conditions of primary cell cultures better and assures a steadier,&#160;more quantifiable extracellular relay of signals to the cells. 
For demonstration purposes, human liver equivalents, generated by aggregating differentiated HepaRG cells with human hepatic stellate cells in hanging drop plates, were cocultured with human skin punch biopsies for up to 28 days inside the microbioreactor. The use of cell culture inserts enables the skin to be cultured at an air-liquid interface, allowing topical substance exposure. The microbioreactor system is capable of supporting these cocultures at near physiologic fluid flow and volume-to-liquid ratios, ensuring stable and organotypic culture conditions. The possibility of long-term cultures enables the repeated exposure to substances. Furthermore, a vascularization of the microfluidic channel circuit using human dermal microvascular endothelial cells yields a physiologically more relevant vascular model.

Here, we present a protocol to coculture primary cells, tissue models and punch biopsies in a microfluidic multi-organ chip for up to 28 days. Human dermal microvascular endothelial cells, liver aggregates and skin biopsies were successfully combined in a common media circulation.

The ever growing amount of new substances released onto the market and the limited predictability of current in vitro test systems has led to a high need ...

Co-culture of Living Microbiome with Microengineered Human Intestinal Villi in a Gut-on-a-Chip Microfluidic Device

Here, we describe a protocol to perform long-term co-culture of multi-species human gut microbiome with microengineered intestinal villi in a human gut-on-a-chip microphysiological device. We recapitulate the intestinal lumen-capillary tissue interface in a microfluidic device, where physiological mechanical deformations and fluid shear flow are constantly applied to mimic peristalsis. In the lumen microchannel, human intestinal epithelial Caco-2 cells are cultured to form a &#39;germ-free&#39; villus epithelium and regenerate small intestinal villi. Pre-cultured microbial cells are inoculated into the lumen side to establish a host-microbe ecosystem. After microbial cells adhere to the apical surface of the villi, fluid flow and mechanical deformations are resumed to produce a steady-state microenvironment in which fresh culture medium is constantly supplied and unbound bacteria (as well as bacterial wastes) are continuously removed. After extended co-culture from days to weeks, multiple microcolonies are found to be randomly located between the villi, and both microbial and epithelial cells remain viable and functional for at least one week in culture. Our co-culture protocol can be adapted to provide a versatile platform for other host-microbiome ecosystems that can be found in various human organs, which may facilitate in vitro study of the role of human microbiome in orchestrating health and disease.

We describe an in vitro protocol to co-culture gut microbiome and intestinal villi for an extended period using a human gut-on-a-chip microphysiological system.

Here, we describe a protocol to perform long-term co-culture of multi-species human gut microbiome with microengineered intestinal villi in a human ...

Dry Film Photoresist-based Electrochemical Microfluidic Biosensor Platform: Device Fabrication, On-chip Assay Preparation, and System Operation

In recent years, biomarker diagnostics became an indispensable tool for the diagnosis of human disease, especially for the point-of-care diagnostics. An easy-to-use and low-cost sensor platform is highly desired to measure various types of analytes (e.g., biomarkers, hormones, and drugs) quantitatively and specifically. For this reason, dry film photoresist technology - enabling cheap, facile, and high-throughput fabrication - was used to manufacture the microfluidic biosensor presented here. Depending on the bioassay used afterwards, the versatile platform is capable of detecting various types of biomolecules. For the fabrication of the device, platinum electrodes are structured on a flexible polyimide (PI) foil in the only clean-room process step. The PI foil serves as a substrate for the electrodes, which are insulated with an epoxy-based photoresist. The microfluidic channel is subsequently generated by the development and lamination of dry film photoresist (DFR) foils onto the PI wafer. By using a hydrophobic stopping barrier in the channel, the channel is separated into two specific areas: an immobilization section for the enzyme-linked assay and an electrochemical measurement cell for the amperometric signal readout.
The on-chip bioassay immobilization is performed by the adsorption of the biomolecules to the channel surface. The glucose oxidase enzyme is used as a transducer for electrochemical signal generation. In the presence of the substrate, glucose, hydrogen peroxide is produced, which is detected at the platinum working electrode. The stop-flow technique is applied to obtain signal amplification along with rapid detection. Different biomolecules can quantitatively be measured by means of the introduced microfluidic system, giving an indication of different types of diseases, or, in regard to therapeutic drug monitoring, facilitating a personalized therapy.

A microfluidic biosensor platform was designed and fabricated using low-cost dry film photoresist technology for the rapid and sensitive quantification of various analytes. This single-use system allows for the electrochemical readout of on-chip-immobilized enzyme-linked assays by means of the stop-flow technique.

In recent years, biomarker diagnostics became an indispensable tool for the diagnosis of human disease, especially for the point-of-care diagnostics. An ...

Immobilization of Live Caenorhabditis elegans Individuals Using an Ultra-thin Polydimethylsiloxane Microfluidic Chip with Water Retention

Radiation is widely used for biological applications and for ion-beam breeding, and among these methods, microbeam irradiation represents a powerful means of identifying radiosensitive sites in living organisms. This paper describes a series of on-chip immobilization methods developed for the targeted microbeam irradiation of live individuals of Caenorhabditis elegans. Notably, the treatment of the polydimethylsiloxane (PDMS) microfluidic chips that we previously developed to immobilize C. elegans individuals without the need for anesthesia is explained in detail. This chip, referred to as a worm sheet, is resilient to allow the microfluidic channels to be expanded, and the elasticity allows animals to be enveloped gently. Also, owing to the self-adsorption capacity of the PDMS, animals can be sealed in the channels by covering the surface of the worm sheet with a thin cover film, in which animals are not pushed into the channels for enclosure. By turning the cover film over, we can easily collect the animals. Furthermore, the worm sheet shows water retention and allows C. elegans individuals to be subjected to microscopic observation for long periods under live conditions. In addition, the sheet is only 300 &#181;m thick, allowing heavy ions such as carbon ions to pass through the sheet enclosing the animals, thus allowing the ion particles to be detected and the applied radiation dose to be measured accurately. Because selection of the cover films used for enclosing the animals is very important for successful long-term immobilization, we conducted the selection of the suitable cover films and showed a recommended one among some films. As an application example of the chip, we introduced imaging observation of muscular activities of animals enclosing the microfluidic channel of the worm sheet, as well as the microbeam irradiation. These examples indicate that the worm sheets have greatly expanded the possibilities for biological experiments.

Immobilization of Live Caenorhabditis elegans Individuals Using an Ultra-thin Polydimethylsiloxane Microfluidic Chip with Water Retention

A series of immobilization methods has been established to allow the targeted irradiation of live Caenorhabditis elegans individuals using a recently developed ultra-thin polydimethylsiloxane microfluidic chip with water retention. This novel on-chip immobilization is also adequate for imaging observations. The detailed treatment and application examples of the chip are explained.

Radiation is widely used for biological applications and for ion-beam breeding, and among these methods, microbeam irradiation represents a powerful means ...

Establishing Single-Cell Based Co-Cultures in a Deterministic Manner with a Microfluidic Chip

Cell co-culture assays have been widely used for studying cell-cell interactions between different cell types to better understand the biology of diseases including cancer. However, it is challenging to clarify the complex mechanism of intercellular interactions in highly heterogeneous cell populations using conventional co-culture systems because the heterogeneity of the cell subpopulation is obscured by the average values; the conventional co-culture systems can only be used to describe the population signal, but are incapable of tracking individual cells behavior. Furthermore, conventional single-cell experimental methods have low efficiency in cell manipulation because of the Poisson distribution. Microfabricated devices are an emerging technology for single-cell studies because they can accurately manipulate single cells at high-throughput and can reduce sample and reagent consumption. Here, we describe the concept and application of a microfluidic chip for multiple single-cell co-cultures. The chip can efficiently capture multiple types of single cells in a culture chamber (~46%) and has a sufficient culture space useful to study the cells&#39; behavior (e.g., migration, proliferation, etc.) under cell-cell interaction at the single-cell level. Lymphatic endothelial cells and oral squamous cell carcinoma were used to perform a single-cell co-culture experiment on the microfluidic platform for live multiple single-cell interaction studies.

This report describes a microfluidic chip-based method to set up a single cell culture experiment in which high-efficiency pairing and microscopic analysis of multiple single cells can be achieved.

Cell co-culture assays have been widely used for studying cell-cell interactions between different cell types to better understand the biology of diseases ...

Speed

Subtitles

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