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 fluidic channels for salt bridges (Figure 2A and 2B).</li>
	</ol>
	</li>
	<li>Scribe an individual pattern on a PMMA sheet or a double-side tape by loading the corresponding file into the CO2 laser scriber.
	<ol>
		<li>Turn on the laser scriber, and check its connection to the computer. Open the pattern to be ablated using the commercial software.</li>
		<li>Place a PMMA sheet or a double-sided tape on top of the stage of the scriber. Adjust the focus of the CO2 laser if necessary using the calibration bar and the visible He-Ne laser.</li>
		<li>Load the pattern into the scriber for ablation on the PMMA sheet or the tape.</li>
		<li>Pick up the patterned sheet or tape, remove unwanted pieces, and clean the surface with nitrogen blowing. 
		NOTE: The thickness of the PMMA sheet is 1 mm, and that of the double-sided tape is 0.07 mm or 0.22 mm.</li>
	</ol>
	</li>
</ol>
2. Chip Assembly
<ol>
	<li>With super glue, attach acrylic adaptors (length x width x height = 10 mm x 10 mm x 5 mm, with screw threads in the middle) to the top-most layer of the chip by aligning the screw threads and the holes on the top-most layer. These adaptors serve as medium inlets/outlets and salt bridge connectors.</li>
	<li>Assemble the microfluidic chip inside a laminar flow hood.</li>
	<li>Assemble the chemical-shear stress microfluidic chip (CSS chip).
	<ol>
		<li>Attach 3 sheets of scribed PMMA sheets using 2 pieces of scribed double-sided tape.&#160;(Figure 1A and 1B).</li>
		<li>Add one more piece of the 0.07 mm thick double sided tape to the bottom of the chip and attach the chip to a 10 cm diameter Petri dish.</li>
		<li>Put the assembly chip in the vacuum chamber for overnight.</li>
	</ol>
	</li>
	<li>To assemble the chemical-electric field chip (CEF chip), attach the PMMA sheet to a double-sided tape of thickness = 0.22 mm (Figure 2A and 2B).
	<ol>
		<li>Attach the scribed PMMA sheet and double-sided tape to a 10 cm diameter Petri dish using this same piece of tape.</li>
	</ol>
	</li>
	<li>Leave the assembled chip inside the hood and expose it to UV for 30 min for sterilization.&#160;</li>
	<li>Connect the inlets to two 3-ml syringes via plastic tubes and finger-tight nuts. Connect the outlet to a waste tube via a plastic tube and a finger-tight nut. 
	NOTE: Autoclave all tubes and nuts at 121 &#176;C for 15 min prior to usage.</li>
	<li>Slowly depress the plunger on the syringe to prime the fluidic channels with PBS. Push syringes back and forth to remove bubbles.</li>
	<li>Put the chips inside an incubator overnight at 37 &#176;C under 5% CO2. 
	NOTE: These two steps are aimed to wash the chip and remove any remaining bubbles within the chip.</li>
	<li>Take the chip out of the incubator.</li>
	<li>Prepare agar salt bridges for generating electric fields in the CEF chip.&#160;
	<ol>
		<li>Dissolve 3% low melting point agarose in 1x phosphate buffered saline (PBS) buffer using a microwave.&#160;</li>
		<li>Inject solution-phased agarose into the salt bridge channel and insert the electrodes before solution solidifies.</li>
		<li>Place silver/silver-chloride electrodes into the tubular nuts.</li>
	</ol>
	</li>
	<li>Connect the syringes to the syringe pumps, and continuously flow the culture medium (Dulbecco&#8217;s Modified Eagle&#8217;s medium, DMEM) into the fluidic channel for 10 min at a flow rate of 20 &#956;l/min to replace PBS.</li>
</ol>
3. Cell Preparation and Experimental Setup
NOTE: Pre-warm 1x PBS, culture medium (DMEM plus FBS), and trypsin in a 37 &#176;C water bath before usage.
<ol>
	<li>Plate 2 x 105 lung cancer CL1-5 cells25,26 in a 10 cm Petri dish supplied with DMEM plus 10% fetal bovine serum (FBS). Incubate the cells inside an incubator under 5% CO2 at 37 &#176;C until 90% confluence.</li>
	<li>Aspirate the medium and wash the cells once with pre-warmed 1x PBS. Add 2 ml of 0.05% trypsin buffer to the cells and wait for 2 to 3 min at 37 &#176;C to detach the cells.</li>
	<li>Transfer the cells to a 15 ml sterile centrifugation tube and add 6 ml of culture medium into the tube. Gently invert the tube for mixing and take out 5 &#181;l of the cell-containing medium for counting the cell number in a hemocytometer.</li>
	<li>Centrifuge the tube at 300 x g for 5 min. Suspend 106 cells in 1 ml of culture medium and place the cell-containing medium in a 1 ml syringe.</li>
	<li>Infuse the cell-containing medium into the microfluidic chip from the outlet and make sure that the solution distributes all through the five culture areas. Incubate the chip inside an incubator under 5% CO2 at 37 &#176;C for 2 hr.&#160;</li>
</ol>
4. Experimental Setup
<ol>
	<li>Take the chip out of the incubator and place it on top of a transparent indium tin oxide (ITO) glass heater. 
	NOTE: The ITO glass is connected to a proportional-integral-derivative (PID) controller for maintaining the temperature at 37 &#177; 0.5 &#176;C via feedback from a thermal coupler clamped tightly between the ITO heater and the chip.</li>
	<li>Put the chip-heater assembly on top of a motorized XY stage of an inverted microscope for tracking cell migration or a fixed XY stage of an inverted fluorescent microscope for measuring the production of ROS.</li>
	<li>To generate different chemical concentrations, fill two syringes with 5 ml of chemical solutions of relative concentrations 0 and 1 (dissolved in culture medium) and pump them into the chip at desired flow rates: in the CSS chip, at a flow rate of 0.3 ml/min for 1 hr; in the CEF chip, at a flow rate of 20 &#181;l/min for the first 20 min and a flow rate of 20 &#181;l/hr for another 120 min (total time = 2 hr).</li>
	<li>In the CEF chip, for tracking cell migration, program the XY stage of the microscope to repeat taking photos, via a digital single-lens reflex (DSLR) camera, of certain field of views (FOVs) within culture areas every 15 min for 2 hr.</li>
	<li>For measuring the production of ROS, use the fluorescence-based indicator 2&#8242;-7&#8242;-dichlorodihydrofluoresce diacetate (2&#8242;-7&#8242;-DCFDA).
	<ol>
		<li>Prepare the stock of 2&#8242;-7&#8242;-DCFDA at 10 mM in molecular biology grade dimethyl sulfoxide (DMSO). Dilute DCFDA in DMEM only without serum (5 &#181;M in DMEM). The potential deacetylase could increase the background signals and decrease the signals in cells.</li>
		<li>After 1 hr of shear stress stimulus in the CSS chip or after 2 hr of EF stimulus in the CEF chip, pump 2&#8242;-7&#8242;-DCFDA (5 &#181;M in DMEM) into the chip at a flow rate of 20 &#181;l/min for the first 20 min and a flow rate of 20 &#181;l/hr for another 20 min. For washing, pump DMEM into the chip at a flow rate of 20 &#181;l/hr for 20 min.</li>
		<li>Take photos, via a charge-coupled device (CCD) camera, of certain FOVs within culture areas for analyzing the fluorescent intensities.</li>
	</ol>
	</li>
</ol>
5. Calculations of Chemical Concentrations, Shear Stresses, and Electric Fields
<ol>
	<li>In both the CSS chip and the CEF chip, calculate the chemical concentrations in the five culture areas. For example, by injecting H2O2 of concentrations 0 and 200 &#181;M from the two inlets, concentrations of 0, 25, 100, 175, and 200 &#181;M are generated. 
	NOTE: By assuming that all liquids split-flow smoothly and equally around the fork, the relative concentrations in the five culture areas are 0, 1/8, 1/2, 7/8, and 1, respectively.</li>
	<li>In the CSS chip, calculate the shear stress (&#964;) within each of the culture areas using <img alt="Equation 1" src="/files/ftp_upload/54397/54397eq1.jpg" /> 27, where Q is the volume flow rate,&#160;&#951; is the fluidic viscosity, h is the height of the channel, and w is the width of the channel. 
	NOTE: By setting Q = 0.3 ml/min in each inlet (Q = 0.12 ml/min in each culture area), &#951; = 0.0008 Pa&#183;s for culture medium, h = 1 mm, and w = 1 ~ 4 mm, the shear stress is calculated to range from 0.0048 Pa (4 mm wide region) to 0.0192 Pa (1 mm wide region).</li>
	<li>In the CEF chip, calculate the EF strength within each of the culture areas using E = I/(&#963;Aeff) (Ohm&#39;s law), where I is the electric current flowing across the fluidic channel, &#963; is the electrical conductivity of the culture medium, and Aeff is the effective cross-sectional area of the channel. 
	NOTE: Using &#963; = 1.38 &#937;-1m-1 for culture medium and Aeff = 0.22 mm2 (width = 1 mm and height = 0.22 mm), the EF strength is calculated to be E (mV/mm) = I (A) &#215; 3.3 &#215; 106.
	<ol>
		<li>As shown in Figure 2D, treat the equivalent circuit as five C-section circuits with four (8 + 35 + 8) segments and one (5 + 35 + 5) segment. 
		NOTE: By analyzing this parallel circuit according to Kirchhoff&#39;s voltage law and Ohm&#39;s law, currents flowing across five culture areas, I1 through I5 from bottom to top, are calculated to be around 0.49I (area 1), 0.25I (area 2), 0.13I (area 3), 0.08I (area 4), and 0.05I (area 5), respectively, where I is the total direct current (dc). With an applied dc of 0.157 mA, EFs of 254, 130, 67, 41, and 26 mV/mm are generated within the five culture areas. 
		NOTE: For a simplified electrical analysis of the microfluidic network, all fluidic segments are considered as resistors with resistance proportional to their lengths.</li>
	</ol>
	</li>
</ol>
6. Data Analysis
Note: Data analysis is performed using the ImageJ software.
<ol>
	<li>Analyze the Production of ROS.
	<ol>
		<li>Run the ImageJ software. Go to &#34;File&#34; &#8594; Open to load a fluorescent image to be analyzed.</li>
		<li>Go to &#34;Image&#34; &#8594; &#34;Type&#34; &#8594; &#34;16-bit&#34; to change the image to a gray scale.</li>
		<li>Draw a polygon to enclose a cell. Go to &#34;Analyze&#34; &#8594; &#34;Measure&#34; to measure the mean fluorescent intensity of the cell.</li>
		<li>Repeat 6.1.3 to collect intensities from at least 50 cells of three independent experiments, and calculate the mean intensity with standard error of mean (SEM).</li>
		<li>Repeat 6.1.1-6.1.4 for each experimental condition.</li>
	</ol>
	</li>
	<li>Analyze Cell Migration.
	<ol>
		<li>Run the ImageJ software. Go to &#34;File&#34; &#8594; Open to load an image taken at time = 0 to be analyzed.</li>
		<li>Draw a polygon to enclose a cell. Go to &#34;Analyze&#34; &#8594; &#34;Measure&#34; to measure the center of mass of the cell as (x1, y1).</li>
		<li>Repeat 6.2.1- 6.2.2 to measure the center of mass of the same cell as (x2, y2) from another image take at time = t.</li>
		<li>Calculate the migration rate (in &#181;m/hr) of this cell as <img alt="Equation 2" src="/files/ftp_upload/54397/54397eq2.jpg" />.</li>
		<li>Repeat 6.2.1-6.2.4 to collect migration rates from at least 50 cells of three independent experiments, and calculate the mean migration rate with standard error of mean (SEM).</li>
		<li>From 6.2.2 and 6.2.3, calculate the migration directedness of this cell as cosine &#952; or <img alt="Equation 3" src="/files/ftp_upload/54397/54397eq3.jpg" />, where &#952; is the angle between the vector of the applied EF (from positive to negative) and the vector from the start to the end position of the cell (Figure 2G).</li>
		<li>Repeat 6.2.6 to collect migration directedness from at least 50 cells of three independent experiments, and calculate the mean migration directedness with standard error of mean (SEM).</li>
		<li>Calculate the mean migration rate with SEM and the mean migration directedness with SEM for each experimental condition. 
		NOTE: A directedness of +1 indicates that all cells migrate toward the cathode, and a -1 value indicates that all cells migrate toward the anode. The directedness of a group of randomly moving cells is close to 0.</li>
	</ol>
	</li>
	<li>Analyze Cell Alignment.
	<ol>
		<li>Run the ImageJ software. Go to &#34;File&#34; &#8594; &#34;Open&#34; to load an image to be analyzed.</li>
		<li>Treat the cell as an ellipse and draw a line to indicate the long axis of a cell. Go to &#34;Analyze&#34;&#160;&#8594; &#34;Measure&#34; to measure the angle &#946; between the line and the horizontal EF direction.</li>
		<li>Repeat 6.3.2 to collect &#946; from at least 50 cells of three independent experiments, and calculate mean cosine &#946; with standard error of mean (SEM).</li>
		<li>Repeat 6.3.1-6.3.3 for each experimental condition. 
		NOTE: A cos&#946; of +1 indicates that all cells align in parallel to the applied EF, and a 0 value indicates that all cells align perpendicularly to the applied EF.</li>
	</ol>
	</li>
</ol>

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Turbulent+fluid+shear+stress+induces+vascular+endothelial+cell+turnover+in+vitro.">Turbulent fluid shear stress induces vascular endothelial cell turnover in vitro.</a> Proc Natl Acad Sci U S A. 83, 2114-2117 (1986).</li><li>Zoro, B. J., Owen, S., Drake, R. A., Hoare, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+impact+of+process+stress+on+suspended+anchorage-dependent+mammalian+cells+as+an+indicator+of+likely+challenges+for+regenerative+medicines.">The impact of process stress on suspended anchorage-dependent mammalian cells as an indicator of likely challenges for regenerative medicines.</a> Biotechnol Bioeng. 99, 468-474 (2008).</li><li>Chin, L. K., 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=Production+of+reactive+oxygen+species+in+endothelial+cells+under+different+pulsatile+shear+stresses+and+glucose+concentrations.">Production of reactive oxygen species in endothelial cells under different pulsatile shear stresses and glucose concentrations.</a> Lab Chip. 11, 1856-1863 (2011).</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></ol>

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><tbody><tr><td>Dulbecco&#039;s Modified Eagle Medium (DMEM)</td><td>Gibco</td><td>11965-092</td><td>Cell culture medium</td></tr><tr><td>Trypsin</td><td>Gibco</td><td>25300-054</td><td>detach cell from the dish</td></tr><tr><td>Fetal bovine serum (FBS)</td><td>Gibco</td><td>10082147</td><td>Cell culture medium</td></tr><tr><td>10 cm cell culture Petri dish</td><td>Nunc</td><td>150350</td><td>Cell culture</td></tr><tr><td>Bright-Line&amp;nbsp;Hemacytometer</td><td>Sigma</td><td>Z359629</td><td>Cell Counting Equipment</td></tr><tr><td>PMMA</td><td>Customized</td><td>Customized</td><td>Microfluidic chip</td></tr><tr><td>Adaptor</td><td>Customized</td><td>Customized</td><td>Microfluidic chip</td></tr><tr><td>0.07/0.22 mm double-sided tape&amp;nbsp;</td><td>3M</td><td>8018/9088</td><td>Microfluidic chip</td></tr><tr><td>Low melting point agarose</td><td>Sigma</td><td>A9414</td><td>Salt bridge</td></tr><tr><td>2&#039;-7&#039;-dichlorodihydrofluoresce diacetate</td><td>Sigma</td><td>D6883</td><td>Intracellular ROS measurement</td></tr><tr><td>Indium tin oxide (ITO) glass</td><td>Merck</td><td>300739</td><td>Heater</td></tr><tr><td>Proportional-integral-derivative controller&amp;nbsp;</td><td>JETEC Electronics Co.</td><td>TTM-J4-R-AB</td><td>Temperature controller</td></tr><tr><td>Thermal coupler</td><td>TECPEL</td><td>TPK-02A</td><td>Temperature controller</td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; laser scriber</td><td>Laser Tools &amp;amp; Technics Corp.</td><td>ILS2</td><td>Microfluidic chip fabrication</td></tr><tr><td>Syringe pumps</td><td>New Era</td><td>NE-300</td><td>Pumping medium and chemicals into the chip</td></tr><tr><td>Power supply</td><td>Major Science&amp;nbsp;</td><td>MP-300V</td><td>Supplying direct currents</td></tr><tr><td>Inverted microscope</td><td>Olympus</td><td>CKX41</td><td>Monitoring cell migration</td></tr><tr><td>Inverted fluorescent microscope</td><td>Nikon</td><td>TS-100</td><td>Monitoring cell migartion and fluorescencent signals</td></tr><tr><td>DSLR camera</td><td>Canon</td><td>60D</td><td>Recording bright-field images&amp;nbsp;</td></tr><tr><td>CCD camera</td><td>Nikon</td><td>DS-Qi1</td><td>Recording fluorescent images&amp;nbsp;</td></tr><tr><td>super glue</td><td>3M Scotch</td><td>7004</td><td>Attaching adaptors to PMMA substrates</td></tr><tr><td>AutoCAD</td><td>Autodesk Inc.</td><td></td><td>Designing microfluidic chips</td></tr><tr><td>DMSO</td><td>Sigma</td><td>D8418</td><td>Dissolving DCFDA</td></tr><tr><td>ImageJ</td><td>National Institutes of Health</td><td></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 ...

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Bioengineering

8.8K Views.  National Taiwan University. 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.

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

Microfabricated Platforms for Mechanically Dynamic Cell Culture

The ability to systematically probe in vitro cellular response to combinations of mechanobiological stimuli for tissue engineering, drug discovery or fundamental cell biology studies is limited by current bioreactor technologies, which cannot simultaneously apply a variety of mechanical stimuli to cultured cells. In order to address this issue, we have developed a series of microfabricated platforms designed to screen for the effects of mechanical stimuli in a high-throughput format. In this protocol, we demonstrate the fabrication of a microactuator array of vertically displaced posts on which the technology is based, and further demonstrate how this base technology can be modified to conduct high-throughput mechanically dynamic cell culture in both two-dimensional and three-dimensional culture paradigms.

In this protocol, we demonstrate the fabrication of a microactuator array of vertically displaced posts on which the technology is based, and how this base technology can be modified to conduct high-throughput mechanically dynamic cell culture in both two-dimensional and three-dimensional culture paradigms.

The ability to systematically probe in vitro cellular response to combinations of mechanobiological stimuli for tissue engineering, drug discovery or ...

Parallel-plate Flow Chamber and Continuous Flow Circuit to Evaluate Endothelial Progenitor Cells under Laminar Flow Shear Stress

The overall goal of this method is to describe a technique to subject adherent cells to laminar flow conditions and evaluate their response to well quantifiable fluid shear stresses1.
Our flow chamber design and flow circuit (Fig. 1) contains a transparent viewing region that enables testing of cell adhesion and imaging of cell morphology immediately before flow (Fig. 11A, B), at various time points during flow (Fig. 11C), and after flow (Fig. 11D). These experiments are illustrated with human umbilical cord blood-derived endothelial progenitor cells (EPCs) and porcine EPCs2,3.
This method is also applicable to other adherent cell types, e.g. smooth muscle cells (SMCs) or fibroblasts.
The chamber and all parts of the circuit are easily sterilized with steam autoclaving. In contrast to other chambers, e.g. microfluidic chambers, large numbers of cells (&gt; 1 million depending on cell size) can be recovered after the flow experiment under sterile conditions for cell culture or other experiments, e.g. DNA or RNA extraction, or immunohistochemistry (Fig. 11E), or scanning electron microscopy5. The shear stress can be adjusted by varying the flow rate of the perfusate, the fluid viscosity, or the channel height and width. The latter can reduce fluid volume or cell needs while ensuring that one-dimensional flow is maintained. It is not necessary to measure chamber height between experiments, since the chamber height does not depend on the use of gaskets, which greatly increases the ease of multiple experiments. Furthermore, the circuit design easily enables the collection of perfusate samples for analysis and/or quantification of metabolites secreted by cells under fluid shear stress exposure, e.g. nitric oxide (Fig. 12)6.

We are describing a method to subject adherent cells to laminar flow shear stress in a sterile continuous flow circuit. The cells' adhesion, morphology can be studied through the transparent chamber, samples obtained from the circuit for metabolite analysis and cells harvested after shear exposure for future experiments or culture.

The overall goal of this method is to describe a technique to subject adherent cells to laminar flow conditions and evaluate their response to well ...

A Microfluidic Device with Groove Patterns for Studying Cellular Behavior

We describe a microfluidic device with microgrooved patterns for studying cellular behavior. This microfluidic platform consists of a top fluidic channel and a bottom microgrooved substrate. To fabricate the microgrooved channels, a top poly(dimethylsiloxane) (PDMS) mold containing the impression of the microfluidic channels was aligned and bonded to a microgrooved substrate. Using this device, mouse fibroblast cells were immobilized and patterned within microgrooved substrates (25, 50, 75, and 100 &mu;m wide). To study apoptosis in a microfluidic device, media containing hydrogen peroxide, Annexin V, and propidium iodide was perfused into the fluidic channel for 2 hours. We found that cells exposed to the oxidative stress became apoptotic. These apoptotic cells were confirmed by Annexin V that bound to phosphatidylserine at the outer leaflet of the plasma membrane during the apoptosis process. Using this microfluidic device with microgrooved patterns, the apoptosis process was observed in real-time and analyzed by using an inverted microscope containing an incubation chamber (37&deg;C, 5% CO2). Therefore, this microfluidic device incorporated with microgrooved substrates could be useful for studying the cellular behavior and performing high-throughput drug screening.

We describe a protocol for the fabrication of microfluidic devices that can enable cell capture and culture. In this approach patterned microstructures such as grooves within microfluidic channels are used to create low shear stress regions within which cell can dock.

We describe a microfluidic device with microgrooved patterns for studying cellular behavior.  This microfluidic platform consists of a top fluidic channel ...

Biology

A Microfluidic Device for Studying Multiple Distinct Strains

The study of cell responses to environmental changes poses many experimental challenges: cells need to be imaged under changing conditions, often in a comparative manner. Multiwell plates are routinely used to compare many different strains or cell lines, but allow limited control over the environment dynamics. Microfluidic devices, on the other hand, allow exquisite dynamic control over the surrounding conditions, but it is challenging to image and distinguish more than a few strains in them. Here we describe a method to easily and rapidly manufacture a microfluidic device capable of applying dynamically changing conditions to multiple distinct yeast strains in one channel. The device is designed and manufactured by simple means without the need for soft lithography. It is composed of a Y-shaped flow channel attached to a second layer harboring microwells. The strains are placed in separate microwells, and imaged under the exact same dynamic conditions. We demonstrate the use of the device for measuring protein localization responses to pulses of nutrient changes in different yeast strains.

We present a simple method to produce microfluidic devices capable of applying similar dynamic conditions to multiple distinct strains, without the need for a clean room or soft lithography.

The study of cell responses to environmental changes poses many experimental challenges: cells need to be imaged under changing conditions, often in a ...

A Microfluidic Technique to Probe Cell Deformability

Here we detail the design, fabrication, and use of a microfluidic device to evaluate the deformability of a large number of individual cells in an efficient manner. Typically, data for ~102 cells can be acquired within a 1 hr experiment. An automated image analysis program enables efficient post-experiment analysis of image data, enabling processing to be complete within a few hours. Our device geometry is unique in that cells must deform through a series of micron-scale constrictions, thereby enabling the initial deformation and time-dependent relaxation of individual cells to be assayed. The applicability of this method to human promyelocytic leukemia (HL-60) cells is demonstrated. Driving cells to deform through micron-scale constrictions using pressure-driven flow, we observe that human promyelocytic (HL-60) cells momentarily occlude the first constriction for a median time of 9.3 msec before passaging more quickly through the subsequent constrictions with a median transit time of 4.0 msec per constriction. By contrast, all-trans retinoic acid-treated (neutrophil-type) HL-60 cells occlude the first constriction for only 4.3 msec before passaging through the subsequent constrictions with a median transit time of 3.3 msec. This method can provide insight into the viscoelastic nature of cells, and ultimately reveal the molecular origins of this behavior.

We demonstrate a microfluidics-based assay to measure the timescale for cells to transit through a sequence of micron-scale constrictions.

Here we detail the design, fabrication, and use of a microfluidic device to evaluate the deformability of a large number of individual cells in an ...

Micropatterning and Assembly of 3D Microvessels

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass based substrates, and hydrogel-based tube formation assays. These assays, while informative, do not recapitulate lumen geometry, proper extracellular matrix, and multi-cellular proximity, which play key roles in modulating vascular function. This manuscript describes an injection molding method to generate engineered vessels with diameters on the order of 100 &#181;m. Microvessels are fabricated by seeding endothelial cells in a microfluidic channel embedded within a native type I collagen hydrogel. By incorporating parenchymal cells within the collagen matrix prior to channel formation, specific tissue microenvironments can be modeled and studied. Additional modulations of hydrodynamic properties and media composition allow for control of complex vascular function within the desired microenvironment. This platform allows for the study of perivascular cell recruitment, blood-endothelium interactions, flow response, and tissue-microvascular interactions. Engineered microvessels offer the ability to isolate the influence from individual components of a vascular niche and precisely control its chemical, mechanical, and biological properties to study vascular biology in both health and disease.

This manuscript presents an injection molding method to engineer microvessels that recapitulate physiological properties of endothelium. The microfluidic-based process creates patent 3D vascular networks with tailorable conditions, such as flow, cellular composition, geometry, and biochemical gradients. The fabrication process and examples of potential applications are described.

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass ...

A Microfluidic Platform for Stimulating Chondrocytes with Dynamic Compression

Mechanical stimuli are known to modulate biological functions of cells and tissues. Recent studies have suggested that compressive stress alters growth plate cartilage architecture and results in growth modulation of long bones of children. To determine the role of compressive stress in bone growth, we created a microfluidic device actuated by pneumatic pressure, to dynamically (or statically) compress growth plate chondrocytes embedded in alginate hydrogel cylinders. In this article, we describe detailed methods for fabricating and characterizing this device. The advantages of our protocol are: 1) Five different magnitudes of compressive stress can be generated on five technical replicates in a single platform, 2) It is easy to visualize cell morphology via a conventional light microscope, 3) Cells can be rapidly isolated from the device after compression to facilitate downstream assays, and 4) The platform can be applied to study mechanobiology of any cell type that can grow in hydrogels.

This article provides detailed methods for fabricating and characterizing a pneumatically actuating microfluidic device for chondrocyte compression.

Mechanical stimuli are known to modulate biological functions of cells and tissues. Recent studies have suggested that compressive stress alters growth ...

Generation of Dynamical Environmental Conditions using a High-Throughput Microfluidic Device

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and organs are either highly expensive, like robotics, or lack nanoliter volume and millisecond time accuracy in liquid manipulation. We herein present the design and fabrication of a microfluidic system, which consists of 1,500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. To demonstrate the capacities of the microfluidic device, neural stem cell (NSC) spheres are maintained in the proposed system. We observed that when the NSC sphere is exposed to CXCL in day 1 and EGF in day 2, the round-shaped conformation is well maintained. Variation in the input order of 6 drugs causes morphological changes to the NSC sphere and the expression level representative marker for NSC stemness (i.e., Hes5 and Dcx). These results indicate that dynamic and complex environmental conditions have great effects on NSC differentiation and self-renewal, and the proposed microfluidic device is a suitable platform for high throughput studies on the complex life machinery.

We present a microfluidic system for high throughput studies on complex life machinery, which consists of 1500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. The microfluidic chip allows for the analysis of the highly complex and dynamic micro-environmental conditions in vivo.

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and ...

Developing 3D Organized Human Cardiac Tissue within a Microfluidic Platform

The leading cause of death worldwide persists as cardiovascular disease (CVD). However, modeling the physiological and biological complexity of the heart muscle, the myocardium, is notoriously difficult to accomplish in vitro. Mainly, obstacles lie in the need for human cardiomyocytes (CMs) that are either adult or exhibit adult-like phenotypes and can successfully replicate the myocardium&#39;s cellular complexity and intricate 3D architecture. Unfortunately, due to ethical concerns and lack of available primary patient-derived human cardiac tissue, combined with the minimal proliferation of CMs, the sourcing of viable human CMs has been a limiting step for cardiac tissue engineering. To this end, most research has transitioned toward cardiac differentiation of human induced pluripotent stem cells (hiPSCs) as the primary source of human CMs, resulting in the wide incorporation of hiPSC-CMs within in vitro assays for cardiac tissue modeling.
Here in this work, we demonstrate a protocol for developing a 3D mature stem cell-derived human cardiac tissue within a microfluidic device. We specifically explain and visually demonstrate the production of a 3D in vitro anisotropic cardiac tissue-on-a-chip model from hiPSC-derived CMs. We primarily describe a purification protocol to select for CMs, the co-culture of cells with a defined ratio via mixing CMs with human CFs (hCFs), and suspension of this co-culture within the collagen-based hydrogel. We further demonstrate the injection of the cell-laden hydrogel within our well-defined microfluidic device, embedded with staggered elliptical microposts that serve as surface topography to induce a high degree of alignment of the surrounding cells and the hydrogel matrix, mimicking the architecture of the native myocardium. We envision that the proposed 3D anisotropic cardiac tissue-on-chip model is suitable for fundamental biology studies, disease modeling, and, through its use as a screening tool, pharmaceutical testing.

The goal of this protocol is to explain and demonstrate the development of a three-dimensional (3D) microfluidic model of highly aligned human cardiac tissue, composed of stem cell-derived cardiomyocytes co-cultured with cardiac fibroblasts (CFs) within a biomimetic, collagen-based hydrogel, for applications in cardiac tissue engineering, drug screening, and disease modeling.

The leading cause of death worldwide persists as cardiovascular disease (CVD). However, modeling the physiological and biological complexity of the heart ...

Electrophysiological Recordings of Single-cell Ion Currents Under Well-defined Shear Stress

Fluid shear stress is well known to play a major role in endothelial function. In most vascular beds, elevated shear stress from acute increases in blood flow triggers a signaling cascade resulting in vasodilation thereby alleviating mechanical stress on the vascular wall. The pattern of shear stress is also well known to be a critical factor in the development of atherosclerosis with laminar shear stress being atheroprotective and disturbed shear stress being pro-atherogenic. While we have a detailed understanding of the various intermediate cell signaling pathways, the receptors that first translate the mechanical stimulus into chemical mediators are not completely understood. Mechanosensitive ion channels are critical to the response to shear and regulate shear-induced cell signaling thereby controlling the production of vasoactive mediators. These channels are among the earliest activated signaling components to shear and have been linked to shear-induced vasodilation through promoting nitric oxide production (e.g., inwardly rectifying K+ [Kir] and transient receptor potential [TRP] channels) and endothelium hyperpolarizing factor (e.g., Kir and calcium-activated K+ [KCa] channels) and shear-induced vasoconstriction through an undetermined mechanism that involves piezo channels. Understanding the biophysical mechanism by which these channels are activated by shear forces (i.e., directly or through a primary mechano-receptor) could provide potential new targets to resolve the pathophysiology associated with endothelial dysfunction and atherogenesis. It is still a major challenge to record flow-induced activation of ion channels in real time using electrophysiology. The standard methods to expose cells to well-defined shear stress, such as the cone and plate rheometer and closed parallel plate flow chamber do not allow real time study of ion channel activation. The goal of this protocol is to describe a modified parallel plate flow chamber that allows real time electrophysiological recording of mechanosensitive ion channels under well-defined shear stress.

The goal of this protocol is to describe a modified parallel plate flow chamber for use in investigating real time activation of mechanosensitive ion channels by shear stress.

Fluid shear stress is well known to play a major role in endothelial function. In most vascular beds, elevated shear stress from acute increases in blood ...