This work was financially supported by the Ministry of Science and Technology of Taiwan under Contract No. MOST 104-2311-B-002-026 (K. Y. Lo), No. MOST 104-2112-M-030-002 (Y. S. Sun), and National Taiwan University Career Development Project (103R7888) (K. Y. Lo). The authors also thank the Center for Emerging Material and Advanced Devices, National Taiwan University, for the use of the cell culture room.

1. Chip Design and Fabrication
<ol>
	<li>Draw patterns to be ablated on PMMA substrates and double-side tapes using commercial software 24.
	<ol>
		<li>To study the effects of chemical concentrations and shear stresses, draw a &#34;Christmas tree&#34; pattern with a varying width at its end in each of the five culture areas (Figure 1A and 1B).</li>
		<li>To study the effects of chemical concentrations and electric fields, draw a &#34;Christmas tree&#34; pattern with two more...

<ol>
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L., 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=Lung+Cancer+A549+Cells+Migrate+Directionally+in+DC+Electric+Fields+With+Polarized+and+Activated+EGFRs.">Lung Cancer A549 Cells Migrate Directionally in DC Electric Fields With Polarized and Activated EGFRs.</a> Bioelectromagnetics. 30, 29-35 (2009).</li><li>Li, 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=Activated+T+lymphocytes+migrate+toward+the+cathode+of+DC+electric+fields+in+microfluidic+devices.">Activated T lymphocytes migrate toward the cathode of DC electric fields in microfluidic devices.</a> Lab Chip. 11, 1298-1304 (2011).</li><li>Lin, 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=Lymphocyte+electrotaxis+in+vitro+and+in+vivo.">Lymphocyte electrotaxis in vitro and in vivo.</a> J Immunol. 181, 2465-2471 (2008).</li><li>Zhang, 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=Electrically+Guiding+Migration+of+Human+Induced+Pluripotent+Stem+Cells.">Electrically Guiding Migration of Human Induced Pluripotent Stem Cells.</a> Stem Cell Rev. , (2011).</li><li>Song, B., 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=Application+of+direct+current+electric+fields+to+cells+and+tissues+in+vitro+and+modulation+of+wound+electric+field+in+vivo.">Application of direct current electric fields to cells and tissues in vitro and modulation of wound electric field in vivo.</a> Nat Protoc. 2, 1479-1489 (2007).</li><li>Sun, Y. 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The authors have nothing to disclose.

PMMA-based chips are fabricated using laser ablation and writing which are cheaper and easier methods when compared to PDMS-based chips which require more complicated soft lithography. After designing a microfluidic chip, the fabrication and assembly can be done within just 5 min. There are some critical steps that attention should be paid to in performing the experiment. The first is the &#34;assembling&#34; issue. The adaptors should be glued properly to the top-most layer of the chip. Glue could leak into the fluidic ...

In vivo cells are surrounded by a variety of biomolecules including extracellular matrix (ECM), carbohydrates, lipids, and other cells. They functionalize by responding to micro-environmental stimuli such as interactions with ECM and responses to chemical gradients of various growth factors. Traditionally, in vitro cell studies are conducted in cell culture dishes where the consumption of cells and reagents is large and cells grow in a static (non-circulating) environment. Recently, micro-fabricated devices integrated with fluidic components have provided an alternative platform for cell studies in a more controllable way. Such devices are capable of creating a precise micro-environment of chemical and physical stimuli while minimizing the consumption of cells and reagents. These microfluidic chips can be made of glass substrates, silicon wafers, polydimethylsiloxane (PDMS) polymers, polymethylmethacrylate (PMMA) substrates, or polyethyleneterephthalate (PET) substrates 1-3. PDMS-based devices are transparent, biocompatible, and permeable to gases, making them suitable for long-term cell culture and studies. PMMA and PET substrates are cheap and easy to be processed using laser ablation and writing.
Microfluidic devices should provide cells with a stable and controllable micro-environment where cells are subject to different chemical and physical stimuli. For example, microfluidic chips are used to study chemotaxis of cells. Instead of traditional methods that employ Boyden chamber and capillary 4,5 these miniaturized fluidic devices can generate precise chemical gradients for studying cells&#39; behaviors 1,6,7. Another example is to study cells&#39; directional migration under electric fields (EFs), a phenomenon named electrotaxis. Electrotactic behaviors of cells were reported to be related to nerve regeneration 8, embryonic development 9, and wound healing 10,11. And many studies have been performed to investigate the electrotaxis of various cell types including cancer cells 12,13, lymphocytes 14,15, leukemia cells 11, and stem cells 16. Conventionally, Petri dishes and cover glasses are used to construct electrotactic chambers for generating EFs 17. Such simple setups pose problems of medium evaporation and imprecise EFs, but they can be overcome by microfluidic devices of enclosed, well-defined fluidic channels 12,18,19.
To systematically study cellular responses under precise, controllable chemical and electrical stimuli, it would be of great use to develop microfluidic devices capable of providing cells with multiple stimuli at the same time. For example, Li et al. reported a PDMS-based microfluidic device for creating single or coexisting chemical gradients and EFs 20. Kao et al. developed a similar microfluidic chip to modulate the chemotaxis of lung cancer cells by EFs 6. Moreover, to increase the throughput, Hou et al. designed and fabricated a PMMA-based multichannel-dual-electric-field chip to provide cells with 8 different combined stimuli, being (2 EF strengths x 4 chemical concentrations)21. To further increase the throughout and add the shear stress stimulus, we developed two PMMA-based microfluidic devices for studying cellular responses under single or coexisting chemical/electrical/shear stress stimuli.
Reported by Lo et al. 22,23, these devices contain five independent cell culture channels subject to continuous fluidic flowing, mimicking the in vivo circulatory system. In the first chip (the chemical-shear stress chip or the CSS chip), five relative concentrations of 0, 1/8, 1/2, 7/8, and 1 are generated in the culture regions, and a shear stress gradient is produced inside each of the five culture areas. In the second chip (the chemical-electric field chip or the CEF chip), by using one single set of electrodes and 2 syringe pumps, five EF strengths are generated in addition to five different chemical concentrations within these culture areas. Numerical calculations and simulations are performed to better design and operate these chips, and lung cancer cells cultured inside these devices are subject to single or coexisting stimuli for observing their responses with respect to the production of reactive oxygen species (ROS), the migration rate, and the migration direction. These chips are demonstrated to be time-saving, high-throughput and reliable devices for investigating how cells respond to various micro-environmental stimuli.

<table border="0" cellpadding="0" cellspacing="0" width="1070">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:279px;">Dulbecco&#39;s Modified Eagle Medium (DMEM)</td>
			<td style="width:213px;">Gibco</td>
			<td style="width:119px;">11965-092</td>
			<td style="width:460px;">Cell culture medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Trypsin</td>
			<td style="width:213px;">Gibco</td>
			<td style="width:119px;">25300-054</td>
			<td>detach cell from the dish</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Fetal bovine serum (FBS)</td>
			<td style="width:213px;">Gibco</td>
			<td style="width:119px;">10082147</td>
			<td>Cell culture medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">10-cm cell culture Petri dish</td>
			<td style="width:213px;">Nunc</td>
			<td style="width:119px;">150350</td>
			<td>Cell culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Bright-Line&#160;Hemacytometer</td>
			<td style="width:213px;">Sigma</td>
			<td>Z359629</td>
			<td>Cell Counting Equipment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">PMMA</td>
			<td style="width:213px;">Customized</td>
			<td style="width:119px;">Customized</td>
			<td>Microfluidic chip</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Adaptor</td>
			<td style="width:213px;">Customized</td>
			<td style="width:119px;">Customized</td>
			<td>Microfluidic chip</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.07/0.22 mm double-sided tape&#160;</td>
			<td>3M</td>
			<td>8018/9088</td>
			<td>Microfluidic chip</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Low melting point agarose</td>
			<td style="width:213px;">Sigma</td>
			<td style="width:119px;">A9414</td>
			<td>Salt bridge</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2&#39;-7&#39;-dichlorodihydrofluoresce diacetate</td>
			<td>Sigma</td>
			<td style="width:119px;">D6883</td>
			<td>Intracellular ROS measurement</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Indium tin oxide (ITO) glass</td>
			<td style="width:213px;">Merck</td>
			<td>300739</td>
			<td>Heater</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Proportional-integral-derivative controller&#160;</td>
			<td>JETEC Electronics Co.</td>
			<td>TTM-J4-R-AB</td>
			<td>Temperature controller</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Thermal coupler</td>
			<td>TECPEL</td>
			<td style="width:119px;">TPK-02A</td>
			<td>Temperature controller</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">CO2 laser scriber</td>
			<td>Laser Tools &#38; Technics Corp.</td>
			<td style="width:119px;">ILS2</td>
			<td>Microfluidic chip fabrication</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Syringe pumps</td>
			<td>New Era</td>
			<td style="width:119px;">NE-300</td>
			<td>Pumping medium and chemicals into the chip</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Power supply</td>
			<td>Major Science&#160;</td>
			<td style="width:119px;">MP-300V</td>
			<td>Supplying direct currents</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Inverted microscope</td>
			<td>Olympus</td>
			<td>CKX41</td>
			<td>Monitoring cell migration</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Inverted fluorescent microscope</td>
			<td style="width:213px;">Nikon</td>
			<td style="width:119px;">TS-100</td>
			<td>Monitoring cell migartion and fluorescencent signals</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DSLR camera</td>
			<td>Canon</td>
			<td>60D</td>
			<td>Recording bright-field images&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CCD camera</td>
			<td>Nikon</td>
			<td style="width:119px;">DS-Qi1</td>
			<td>Recording fluorescent images&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">super glue</td>
			<td style="width:213px;">3M Scotch</td>
			<td style="width:119px;">7004</td>
			<td>Attaching adaptors to PMMA substrates</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">AutoCAD</td>
			<td style="width:213px;">Autodesk Inc.</td>
			<td style="width:119px;"></td>
			<td>Designing microfluidic chips</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">DMSO</td>
			<td style="width:213px;">Sigma</td>
			<td style="width:119px;">D8418</td>
			<td>Dissolving DCFDA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">ImgeJ</td>
			<td style="width:213px;">National Institutes of Health</td>
			<td style="width:119px;"></td>
			<td>Quantifying fluorescent intensities and cell migration</td>
		</tr>
	</tbody>
</table>

designing microfluidic devices for studying cellular responses under single or coexisting chemical/electrical/shear stress stimuli

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

The Chemical-shear Stress (CSS) Chip
The CSS chip is made of three PMMA sheets, each of thickness 1 mm, attached together via two double-sided tapes, each of thickness 0.07 mm (Figure 1A and 1B). The &#34;Christmas tree&#34; structure generates five relative concentrations of 0, 1/8, 1/2, 7/8, and 1 in the five culture areas. By designing the culture area as a triangle, a shear stress gradient, ...

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

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

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

The overall goal of this procedure is to design and fabricate microfluidic devices for observing how cells respond to environmental stimuli.This method can help answer key questions in the cell biology and biomedical engineering.Such as how cells responses under different environmental stimuli including chemicals, shear stresses, and electric field.The main advantage of this technique is that tubes are easily to be fabricated and assembled.And this these microfludic devices may make the in-vivo condition with high superb capabilities.Demonstrating the procedure will be Tzu-Yuan Chou, an undergraduate student from my lab, and Hsien-San Hou, a post-staff from Dr.Julian Chung's lab.To begin, turn on the carbon dioxide laser scriber and check it's connection to the computer.Then load up a pre-drawn Christmas tree pattern, like one of the patterns shown here into the scriber.Next, place a PMMA sheet or piece of double-sided tape on top of the stage of the scriber.Adjust the focus of the carbon dioxide laser using the calibration bar and the visible helium neon laser.Then scribe the pattern onto the substrate.When finished, pick up the pattern substrate, remove the unwanted pieces, and use a stream of nitrogen to clean the surface of any remaining debris.Repeat this process for each component of the chip.Assemble the chemical shear stress microfluidic chip.To accomplish this, attach three sheets of the chemical shear stress patterned PMMA sheets using two pieces of scribed double-sided tape.Use super glue to attach a acrylic adapters with screw threads in the middle to the top-most PMMA layer of the chip by aligning the screw threads and the holes.These adapters will serve as medium inlets outlets and salt bridge connectors.Then add one more piece of the 07 mm thick double-sided tape to the bottom of the chip, and attach the chip to a 10 cm diameter Petri dish.Once assembled, put the chip in a vacuum chamber for overnight.Now assemble the chemical-electric field chip.Attach the scribed PMMA sheet for the chemical-electric field chip with double-sided tape to a 10 cm diameter Petri dish.Prior to using the chips, expose them to UV for 30 minutes to sterilize them.Next, connect the inlets of the chips to 3 ml PBS-filled syringes via plastic tubes and finger-tight nuts.In the same manner, connect the outlets to a waste tube.Slowly depress the plunger on the syringe to prime the fluidic channels with PBS.Push the syringe back and forth to remove bubbles, then put the chips inside an incubator overnight.To setup the chemical-electric field chips, fill 3%agarous in in the tubular nuts to setup the salt bridges.Then place the silver silver chloride electrodes into the tubular nuts.Grow lung cancer CL1-5 cells in D-MEM supplemented with 10%FBS until they reach 90%confluence.Then aspirate the medium.Wash the cells once with pre-warmed PBS.And add 2 ml of a 05%tripsin buffer solution to a 10 cm diameter plate.Place the cells back at 37

designing-microfluidic-devices-for-studying-cellular-responses-under

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JoVE Journal

Bioengineering

8.8K Görüntüleme.  National Taiwan University. The overall goal of this procedure is to design and fabricate microfluidic devices for observing how cells respond to environmental stimuli.This method can help answer key questions in the cell biology and biomedical engineering.Such as how cells responses under different environmental stimuli including chemicals, shear stresses, and electric field.The main advantage of this technique is that tubes are easily to be fabricated and assembled.And this these microfludic devices may make the in-vi...