This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIP) (No. NRF-2017R1E1A1A01075103), Korea University Grant, and the BK 21 Plus program. It was also supported by the National Institutes of Health (U01CA202123, PO1HL120839, T32HL007118, R01EY019696).

NOTE: Lithography of SU-8 molds for stencils (thickness = 250 &#956;m) and microchannel parts (thickness = 150 &#956;m), glass etching (depth = 100 &#956;m), and cast fabrication were outsourced by sending designs using computer-aided design software to manufacturers.
1. Fabrication of polydimethylsiloxane (PDMS) stencil and microchannel
<ol>
	<li>Design the micropattern of stencil and microchannel.</li>
	<li>Fabricate or outsource SU-8 molds (thickness of ~250 &#956;m for stencils and ~150 &#956;m for microchannels) on silicon wafers (4&#34;&#160;diameter).</li>
	<li>Prepare PDMS mixture by mixing the base elastomer and curing agent at a ratio of 10:1.
	<ol>
		<li>Place 15 mL of the base elastomer in a 50 mL conical tube and add 1.5 mL of curing agent. Prepare two of these.</li>
		<li>Vortex the PDMS mixture for 5 min. Centrifuge the PDMS mixture at 196 x g for 1 min to remove bubbles.</li>
	</ol>
	</li>
	<li>To fabricate PDMS stencil, pour ~1 mL of the PDMS mixture on the wafer, while avoiding SU-8 patterned regions so that the PDMS touches the side of the SU-8 pillars but not the top of the SU-8 pattern.
	<ol>
		<li>Place the wafer on a flat surface for over 30 min at room temperature (RT). Cure the PDMS in a dry oven at 80 &#176;C for over 2 h.</li>
		<li>Carefully peel off the PDMS from SU-8 mold and trim the thin PDMS membrane using a 14 mm hollow punch. Remove the dust on the surface of the PDMS pieces using sticky tape and autoclave the PDMS stencils.</li>
	</ol>
	</li>
	<li>To fabricate PDMS microchannel, pour ~30 mL of PDMS mixture over the SU-8 mold.
	<ol>
		<li>Degas for 30 min in a vacuum chamber and cure the PDMS in a dry oven at 80 &#176;C for over 2 h.</li>
		<li>Carefully peel off the PDMS from SU-8 mold and cut the PDMS to a size of 24 mm x 24 mm. In each PDMS block, create one outlet and three inlets using a 1 mm biopsy punch.</li>
	</ol>
	</li>
</ol>
2. Preparation of bottom glass with polyacrylamide (PA) gel
<ol>
	<li>Manufacture or outsource rectangular slide glasses (24 mm x 24 mm x 1 mm) with a rectangular micro-well (6 mm x 12 mm, 100 &#956;m depth29) by cutting and etching glasses.</li>
	<li>Silanize the surface of a bottom glass30.
	<ol>
		<li>Prepare a bind silane solution by mixing 200 mL of deionized water (DIW), 80 &#956;L of acetic acid, and 50 &#181;L of 3-(trimethoxysilyl) propyl methacrylate (TMSPMA) for 1 h. 
		CAUTION: TMSPMA is a combustible liquid. Follow the recommendations in material safety data sheets. Use only in a chemical fume hood.</li>
		<li>Remove the dust from the surface of the glass by sticky tape and autoclave the glass.</li>
		<li>Cover the etched surface of the bottom glass with 100 &#956;L of the bind silane solution and leave the glass at RT for 1 h.</li>
		<li>Rinse the glass with DIW 3x and let the glass dry at ambient air temperature or by blowing air.</li>
	</ol>
	</li>
	<li>Prepare a gel solution for the PA gel31.
	<ol>
		<li>Prepare a fresh solution of 0.5 % (w/v) ammonium persulfate (APS) by dissolving 5 mg of APS in 1 mL of DIW.</li>
		<li>Prepare the PA gel solution consisting of 138 &#181;L of 40% acrylamide solution, 101 &#181;L of 2% bis-acrylamide solution, 5 &#181;L of fluorescent particle solution (0.2 &#956;m), and 655 &#181;L of DIW. 
		CAUTION: Acrylamide and bisacrylamide solutions are toxic. Wear protective gloves, clothing, and eye protection. Protect the PA gel solution containing fluorescent particles from light. 
		NOTE: Vortex the fluorescent bead solution before pipetting to obtain a uniform number of fluorescent beads per batch.</li>
		<li>After adding 100 &#956;L of the APS solution and 1 &#956;L of tetramethylethylenediamine (TEMED), transfer 10 &#181;L of mixed gel solution onto the rectangular micro-well, and place on top a circular coverslip (18 mm). 
		NOTE: To fill the bottom glass with PA gel without bubbles, place sufficient gel solution on the groove of the bottom glass, carefully slide the coverslip over the gel solution and remove any excess gel solution. 
		CAUTION: The gel is slowly cured for about 40 min. The following procedure for centrifuging gels should be carried out as soon as possible.</li>
		<li>Flip the assembly of custom glass, gel solution, and coverslip, then centrifuge for 10 min at 96 x g to bring fluorescent particles to the top layer of PA gel.</li>
		<li>Remove the assembly from the centrifuge and place it on the flat surface with the coverslip facing down. 
		NOTE: Beginning with step 2.3.6, handle samples in a biosafety cabinet.</li>
		<li>After 30 min, flip the assembly and place it in a 35 mm Petri dish, fill with 2 mL of DIW, and (using forceps) gently remove the coverslip by sliding it to one side. 
		NOTE: Cured PA gel can be stored in DIW for 1 month. However, once collagen is coated on the PA gel, it should be used in an experiment within 1 day.</li>
	</ol>
	</li>
	<li>Coat collagen on the PA gel.
	<ol>
		<li>Dissolve 1 mg/mL sulfosuccinimidyl 6-(4&#39;-azido-2&#39;-nitrophenylamino) hexanoate (sulfo-SANPAH) in warm 50 mM HEPES buffer. Drop 200 &#181;L of the solution onto the gel surface and activate by UV light (365 nm wavelength) for 10 min. 
		NOTE: Protect the sulfo-SANPAH from light.</li>
		<li>Rinse the gel with 0.1 M [4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; HEPES] buffer 2x and with PBS 1x.</li>
		<li>Coat the PA gel with collagen solution (100 &#956;g/mL in PBS, rat tail collagen type I) at 4 &#176;C overnight. On the following day, wash with PBS 3x.</li>
	</ol>
	</li>
</ol>
3. Micropatterning of cell islands
<ol>
	<li>Prepare F-127 (Table of Materials) solution [2% (w/v) in PBS] and immerse the autoclaved PDMS stencil in the F-127 solution. Keep it in a 37 &#176;C incubator for 1 h.</li>
	<li>Prepare cell solution (2 x 106 cell/mL) in cell culture media consisting of: Dulbecco&#8217;s modified Eagle&#8217;s medium (DMEM) with 10% fetal bovine serum (FBS) and 1% antibiotic-antimycotic (AA).</li>
	<li>Wash the PDMS stencil with PBS 3x and remove liquid from both the PDMS stencil and PA gel. Place the PDMS stencil on the PA gel and add PBS to the stencil.</li>
	<li>Remove bubbles in the holes of PDMS stencil by pipetting gently. After removing bubbles, remove PBS from the surface of the PDMS stencil.</li>
	<li>After putting 200 &#181;L of cell solution on PDMS stencil, keep the PA gel in an incubator for 1 h so that cells attach to the PA gel.</li>
	<li>Gently wash off cell solution with cell culture media, remove the PDMS stencil, and add more cell culture media. Check the formation of cell islands under a microscope.</li>
</ol>
4. Assembly of PA gel with PDMS microchannel
<ol>
	<li>Remove dust on the PDMS microchannel using sticky tape, then autoclave.</li>
	<li>Treat the surface of the PDMS microchannel with oxygen plasma (80 W, 50 kHz) for 30 s.</li>
	<li>After removing any fluid on the PA gel-filled bottom glass, place the PDMS microchannel on top of the bottom glass and put the assembly on the custom glass holder.</li>
	<li>Fill the microchannel with cell culture medium. 
	CAUTION: Make sure to remove all the bubbles trapped in the channels by gently flushing warm media with a pipette. Also, while removing bubbles, make sure not to detach micropatterned cell islands.</li>
</ol>
5. Integrated microfluidic system
<ol>
	<li>Connect the tubings.
	<ol>
		<li>Prepare connectors by trimming the tip of needles (18 G) and bending it 90&#176;.</li>
		<li>Prepare tubing lines for three inlets and one outlet.
		<ol>
			<li>For inlet tubing, connect the trimmed needle and a 30 cm mini-volume line with a three-way stopcock. Prepare three of these.</li>
			<li>For outlet tubing, connect the trimmed needle and a 75 cm mini-volume line with a three-way stopcock. Prepare one of these.</li>
			<li>Fill the tubing lines with the medium that has been preheated for 1 h. 
			CAUTION: Make sure to remove all the bubbles trapped in the tubing lines by gently flushing warm media with a syringe.</li>
		</ol>
		</li>
		<li>Prepare reservoirs by removing plungers from syringes and connecting inlet tubing lines.</li>
		<li>Plug the needle connectors of each tubing line into the three inlets and one outlet of the microfluidic device.</li>
	</ol>
	</li>
	<li>Fill the reservoirs with 3 mL of fresh medium or conditioned medium each.
	<ol>
		<li>For the gradient test, fill the left inlet reservoir with 20 ng/mL HGF in the cell culture medium.</li>
		<li>For the visualization of concentration gradient, add a 200 &#181;g/mL fluorescent dye (rhodamine B-dextran, 70 kDa) to the left inlet reservoir.</li>
		<li>Connect the outlet tubing line to a syringe pump. 
		NOTE: The operating mode of the syringe pump is &#34;withdrawal&#34;. Flow rate is changed according to the capacity of the syringe and operating speed of the syringe pump.</li>
	</ol>
	</li>
	<li>Place the integrated microfluidic system on the stage of a conventional epifluorescent microscope. 
	CAUTION: To generate a gentle gradient of HGF in the microfluidic channel, the flow rate should be as slow as 0.1 &#181;L/min. This is sufficiently sensitive so that it requires stabilization for 2 h, and care must be taken to avoid physical disturbance during the experiment.</li>
</ol>
6. Image acquisition
<ol>
	<li>Take images every 10 min for up to 24 h using an automated microscope housed in an incubator. At each timepoint, take a set of images using a 4x objective lens in three different channels, including phase image to visualize cell migration, green fluorescent image to visualize fluorescent beads embedded in PA gel, and red fluorescent image to visualize the concentration gradient of a chemical. &#160;</li>
	<li>After taking time-lapse images, infuse 0.25% trypsin-EDTA solution into microchannels to detach cells from the PA gel. After completely removing cells from the gel, take a green fluorescent image to be used as a reference image for traction microscopy.</li>
</ol>
7. Data analysis
NOTE: A custom code for data analysis was developed using MATLAB, and details have been described elsewhere32,33,34,35,36.
<ol>
	<li>For phase-image analysis, calculate displacements in two consecutive phase images using particle image velocimetry36.</li>
	<li>For Fourier transform traction microscopy, compare each green fluorescent image with the reference image and calculate the displacements in each image using particle image velocimetry36. From the displacements, recover traction made by cells on PA gel using Fourier transform traction microscopy33,34,37.</li>
	<li>From the traction data, calculate stress within the monolayer of the cell island using monolayer stress microscopy based on finite element methods32,34,38.</li>
</ol>

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M., Butler, J. P., Chen, J., Wang, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+and+temporal+traction+response+in+human+airway+smooth+muscle+cells.">Spatial and temporal traction response in human airway smooth muscle cells.</a> American Journal of Physiology - Cell Physiology. 283 (4), 1254-1266 (2002).</li><li>Butler, J. P., Tolic-Norrelykke, I. M., Fabry, B., Fredberg, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Traction+fields,+moments,+and+strain+energy+that+cells+exert+on+their+surroundings.">Traction fields, moments, and strain energy that cells exert on their surroundings.</a> American Journal of Physiology - Cell Physiology. 282 (3), 595-605 (2002).</li><li>Tambe, D. T., 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=Monolayer+stress+microscopy:+limitations,+artifacts,+and+accuracy+of+recovered+intercellular+stresses.">Monolayer stress microscopy: limitations, artifacts, and accuracy of recovered intercellular stresses.</a> PLoS ONE. 8 (2), 55172 (2013).</li><li>Dembo, M., Wang, Y. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stresses+at+the+cell-to-substrate+interface+during+locomotion+of+fibroblasts.">Stresses at the cell-to-substrate interface during locomotion of fibroblasts.</a> Biophysical Journal. 76 (4), 2307-2316 (1999).</li><li>Wang, N., 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=Cell+prestress.+I.+Stiffness+and+prestress+are+closely+associated+in+adherent+contractile+cells.">Cell prestress. I. Stiffness and prestress are closely associated in adherent contractile cells.</a> American Journal of Physiology-Cell Physiology. 282 (3), 606-616 (2002).</li><li>Notbohm, 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=Cellular+Contraction+and+Polarization+Drive+Collective+Cellular+Motion.">Cellular Contraction and Polarization Drive Collective Cellular Motion.</a> Biophysical Journal. 110 (12), 2729-2738 (2016).</li><li>Sunyer, R., 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=Collective+cell+durotaxis+emerges+from+long-range+intercellular+force+transmission.">Collective cell durotaxis emerges from long-range intercellular force transmission.</a> Science. 353 (6304), 1157-1161 (2016).</li><li>Kim, J. H., 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=Propulsion+and+navigation+within+the+advancing+monolayer+sheet.">Propulsion and navigation within the advancing monolayer sheet.</a> Nature Materials. 12 (9), 856-863 (2013).</li></ol>

The authors declare that they have no competing financial interests.

Collective migration of constituent cells is an important process during development and regeneration, and the migrating direction is often guided by the chemical gradient of growth factors4,23. During collective migration, cells keep interacting with neighboring cells and underlying substrates. Such mechanical interactions give rise to emergent phenomena such as durotaxis42, plithotaxis33, and kenotaxis<sup class="...

Cellular migration in biological systems is a fundamental phenomenon involved in tissue formation, the immune response, and wound healing1,2,3. Cellular migration is also an important process in some diseases like cancer4. Cells often migrate as a group rather than individually, which is known as collective cell migration4,5. For cells to move collectively, sensing of the microenvironment is essential6. For instance, cells perceive physicochemical stimuli and respond by changing motility, cell-substrate interactions, and cell-cell interactions, resulting in directional migration along a chemical gradient7,8,9,10. Based on this connection, rapid advancements have been made in lab-on-a-chip technologies that can create well-controlled chemical microenvironments such as the gradient of a chemoattractant11,12,13. While these lab-on-a-chip-based microfluidics have previously been used to study chemotaxis of the cellular ensemble or cellular spheroids14,15,16,17, they have been used mostly in the context of single-cell migration18,19,20,21. Mechanisms underlying a cellular collective response to a chemical gradient is still not well-understood14,22,23,24,25,26. Thus, the development of a platform that enables the spatiotemporal control of soluble factors as well as in situ observation of cells&#39;&#160;biophysical will help to unravel the mechanisms behind collective cell migration.
Developed and described here is a multi-channeled microfluidic system that enables the generation of a concentration gradient of soluble factors that modulates migration of patterned cell clusters. In this study, hepatocyte growth factor (HGF) is chosen to regulate the migratory behavior of Madin-Darby canine kidney (MDCK) cells. HGF is known to attenuate cell-cell integrity and enhance the motility of cells27,28. In the microfluidic system, Fourier transform traction microscopy and monolayer stress microscopy are also incorporated, which allows analysis of the motility, contractile force, and intercellular tension induced by constituent cells in response to an HGF gradient. Within the same island, cells located near the higher concentration of HGF migrate faster and show lower intercellular stress levels than those on the side with lower HGF concentration. The results suggest that this new experimental system is suitable to explore other questions in fields involving collective cellular migration under chemical gradients of various soluble factors.

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			<td height="22" style="height:22px;width:529px;">0.25% trypsin-EDTA (1X)</td>
			<td style="width:164px;">Gibco</td>
			<td style="width:129px;">25200-056</td>
			<td style="width:639px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:529px;">1 M HEPES buffer solution</td>
			<td>Gibco</td>
			<td>15630-056</td>
			<td style="width:639px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">1 mm Biopsy punch</td>
			<td>Integra Miltex</td>
			<td>33-31AA-P/25</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">100 mm petri dishes</td>
			<td>SPL</td>
			<td>10100</td>
			<td style="width:639px;">100 mm diameter, 15 mm height</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">14 mm hollow punch</td>
			<td>ILJIN</td>
			<td>124-0571</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">18 mm &#216; Coverslip</td>
			<td>Marienfeld-Superior</td>
			<td>111580</td>
			<td style="width:639px;">Circular 18 mm, thickness No. 1 (0.13 to 0.16 mm)</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:529px;">2% bis-acrylamide solution</td>
			<td>Bio-Rad</td>
			<td>1610142</td>
			<td style="width:639px;">Wear protective gloves, clothing, and eye protection.</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">3-(Trimethoxysilyl)propyl methacrylate (TMSPMA)</td>
			<td>Sigma-Aldrich</td>
			<td>440159-500ML</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">3-way stopcock</td>
			<td>Hyupsung</td>
			<td>HS-T-61N</td>
			<td style="width:639px;">CAUTION: do not use if previously opened. do not resterlize or resuse</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:529px;">30 cm minimum volume line (for pediatric)</td>
			<td>Hyupsung</td>
			<td>HS-MV-30</td>
			<td style="width:639px;">CAUTION: do not use if previously opened. do not resterlize or resuse</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">35 mm cell culture dish</td>
			<td>Corning</td>
			<td>430165</td>
			<td style="width:639px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">40% Acrylamide Solution</td>
			<td>Bio-Rad</td>
			<td>1610140</td>
			<td style="width:639px;">Wear protective gloves, clothing, and eye protection.</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:529px;">75 cm minimum volume line (for pediatric)</td>
			<td>Hyupsung</td>
			<td>HS-MV-75</td>
			<td style="width:639px;">CAUTION: do not use if previously opened. do not resterlize or resuse</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">acetic acid</td>
			<td>J.T. Baker</td>
			<td>JT9508-03</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Ammonium persulfate (APS)</td>
			<td>Bio-Rad</td>
			<td>1610700</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Antibiotic-Antimycotic</td>
			<td>Gibco</td>
			<td>15240-062</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Bottom glass chip</td>
			<td>MicroFIT</td>
			<td></td>
			<td>24 x 24 x 1 mm, custom-made, rectangular groove (6 x 12 mm, depth : 100 &#956;m)</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Collagen typeI, Rat tail</td>
			<td>Corning</td>
			<td>354236</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Custom glass holder</td>
			<td colspan="2">Han-Gug Mechatronics</td>
			<td>custom-made</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM)</td>
			<td>Welgene</td>
			<td>LM 001-11</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Dulbecco&#39;s Phosphate Buffered Saline (PBS)</td>
			<td>Biowest</td>
			<td>L0615-500</td>
			<td>w/o Magnesium, Calcium</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Fetal bovine serum (FBS)</td>
			<td>Gibco</td>
			<td>26140-179</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:529px;">FluoSpheres amine-modified microspheres</td>
			<td>Invitrogen</td>
			<td>F8764</td>
			<td style="width:639px;">0.2 &#181;m, yellow-green fluorescent(505/515)</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Hepatocyte Growth Factor (HGF)</td>
			<td>Sigma-Aldrich</td>
			<td>H1404-5UG</td>
			<td>recombinant, human</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">JuLI stage live cell imaging system</td>
			<td>NanoEnTek In</td>
			<td></td>
			<td style="width:639px;">Automated X-Y-Z stage and fluorsent imaging Incubator-compatible (37 &#176;C and 5% CO2)</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Madin-Darby Canine Kidney (MDCK) cell</td>
			<td></td>
			<td></td>
			<td>type II</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Oxygen plasma system</td>
			<td>Femto Science</td>
			<td>CUTE-MPR</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Pluronic F-127</td>
			<td>Sigma-Aldrich</td>
			<td>P2443-250G</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Rhodamine B isothiocyanate&#8211;dextran</td>
			<td>Sigma-Aldrich</td>
			<td>R9379-100MG</td>
			<td>70 kDa, used to estimate spatiotemporal distribution of HGF in the microfluidic channel</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Steril hypodermic needle 18 G</td>
			<td>KOVAX</td>
			<td></td>
			<td>Trim the tip of the needle and bend it 90 degrees for connecting in/out ports with volume line</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Sticky tape</td>
			<td>3M/Scotch</td>
			<td>810D</td>
			<td>33 m x 19 mm</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">SU-8 master molds</td>
			<td>MicroFIT</td>
			<td></td>
			<td>4&#8221; diameter, custom-made</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">sulfosuccinimidyl 6-(4&#8217;-azido-2&#8217;-nitrophenylamino)hexanoate (Sulfo-SANPAH)</td>
			<td>Thermo Scientific</td>
			<td>22589</td>
			<td>Store at -20&#176;C. Store protected from moisture and light.</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Sylgard 184 Elastomer Kit</td>
			<td>Dow Corning</td>
			<td></td>
			<td>PDMS</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Syringe pump</td>
			<td>Chemyx Inc.</td>
			<td>model fusion 720</td>
			<td>withdraw fluid</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Syringes</td>
			<td>KOVAX</td>
			<td></td>
			<td>1, 3, 5, 10, or 50 cc for using inlet reservoir or outlet syringe pump</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:529px;">tetramethylethylenediamine (TEMED)</td>
			<td>Bio-Rad</td>
			<td>1610800</td>
			<td style="width:639px;">Wear protective gloves, clothing, and eye protection.</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Ultraviolet (UV) lamp</td>
			<td>UVP LLC</td>
			<td>95-0248-02</td>
			<td style="width:639px;">365 nm wavelength</td>
		</tr>
	</tbody>
</table>

traction microscopy integrated with microfluidics for chemotactic collective migration

Cells change migration patterns in response to chemical stimuli, including the gradients of the stimuli. Cellular migration in the direction of a chemical gradient, known as chemotaxis, plays an important role in development, the immune response, wound healing, and cancer metastasis. While chemotaxis modulates the migration of single cells as well as collections of cells in vivo, in vitro research focuses on single-cell chemotaxis, partly due to the lack of the proper experimental tools. To fill that gap, described here is a unique experimental system that combines microfluidics and micropatterning to demonstrate the effects of chemical gradients on collective cell migration. Furthermore, traction microscopy and monolayer stress microscopy are incorporated into the system to characterize changes in cellular force on the substrate as well as between neighboring cells. As proof-of-concept, the migration of micropatterned circular islands of Madin-Darby canine kidney (MDCK) cells is tested under a gradient of hepatocyte growth factor (HGF), a known scattering factor. It is found that cells located near the higher concentration of HGF migrate faster than those on the opposite side within a cell island. Within the same island, cellular traction is similar on both sides, but intercellular stress is much lower on the side of higher HGF concentration. This novel experimental system can provide new opportunities to studying the mechanics of chemotactic migration by cellular collectives.

To explore collective migration under a chemical gradient, a microfluidic system was integrated with traction microscopy (Figure 1). To build the integrated system, polyacrylamide (PA) gel was cast on custom-cut glass, and MDCK cells were seeded within micropatterned islands made by a PDMS stencil. For this experiment, twelve islands of MDCK cells (four rows by three columns, diameter of ~700 &#956;m) were created. After cells attached to PA gels, the PDMS stencil was removed to initiate col...

Watch this Scientific Journal Video about Traction Microscopy Integrated with Microfluidics for Chemotactic Collective Migration at JoVE.com

Traction Microscopy Integrated with Microfluidics for Chemotactic Collective Migration

Collective cell migration in development, wound healing, and cancer metastasis is often guided by the gradients of growth factors or signaling molecules. Described here is an experimental system combining traction microscopy with a microfluidic system and a demonstration of how to quantify the mechanics of collective migration under biochemical gradient.

Cells change migration patterns in response to chemical stimuli, including the gradients of the stimuli. Cellular migration in the direction of a chemical ...

traction-microscopy-integrated-with-microfluidics-for-chemotactic

Research

JoVE Journal

Bioengineering

6.9K Views.  Korea University. Collective cell migration in development, wound healing, and cancer metastasis is often guided by the gradients of growth factors or signaling molecules. Described here is an experimental system combining traction microscopy with a microfluidic system and a demonstration of how to quantify the mechanics of collective migration under biochemical gradient.

Video: Traction Microscopy Integrated with Microfluidics for Chemotactic Collective Migration

Endothelialized Microfluidics for Studying Microvascular Interactions in Hematologic Diseases

Advances in microfabrication techniques have enabled the production of inexpensive and reproducible microfluidic systems for conducting biological and biochemical experiments at the micro- and nanoscales 1,2. In addition, microfluidics have also been specifically used to quantitatively analyze hematologic and microvascular processes, because of their ability to easily control the dynamic fluidic environment and biological conditions3-6. As such, researchers have more recently used microfluidic systems to study blood cell deformability, blood cell aggregation, microvascular blood flow, and blood cell-endothelial cell interactions6-13.However, these microfluidic systems either did not include cultured endothelial cells or were larger than the sizescale relevant to microvascular pathologic processes. A microfluidic platform with cultured endothelial cells that accurately recapitulates the cellular, physical, and hemodynamic environment of the microcirculation is needed to further our understanding of the underlying biophysical pathophysiology of hematologic diseases that involve the microvasculature.
Here, we report a method to create an "endothelialized" in vitro model of the microvasculature, using a simple, single mask microfabrication process in conjunction with standard endothelial cell culture techniques, to study pathologic biophysical microvascular interactions that occur in hematologic disease. This "microvasculature-on-a-chip" provides the researcher with a robust assay that tightly controls biological as well as biophysical conditions and is operated using a standard syringe pump and brightfield/fluorescence microscopy. Parameters such as microcirculatory hemodynamic conditions, endothelial cell type, blood cell type(s) and concentration(s), drug/inhibitory concentration etc., can all be easily controlled. As such, our microsystem provides a method to quantitatively investigate disease processes in which microvascular flow is impaired due to alterations in cell adhesion, aggregation, and deformability, a capability unavailable with existing assays.

A method to culture an endothelial cell monolayer throughout the entire inner 3D surface of a microfluidic device with microvascular-sized channels (&lt;30 &mu;m) is described. This in vitro microvasculature model enables the study of biophysical interactions between blood cells, endothelial cells, and soluble factors in hematologic diseases.

Advances in  microfabrication techniques have enabled the production of inexpensive and  reproducible microfluidic systems for conducting biological and ...

Creating Adhesive and Soluble Gradients for Imaging Cell Migration with Fluorescence Microscopy

Cells can sense and migrate towards higher concentrations of adhesive cues such as the glycoproteins of the extracellular matrix and soluble cues such as growth factors. Here, we outline a method to create opposing gradients of adhesive and soluble cues in a microfluidic chamber, which is compatible with live cell imaging. A copolymer of poly-L-lysine and polyethylene glycol (PLL-PEG) is employed to passivate glass coverslips and prevent non-specific adsorption of biomolecules and cells. Next, microcontact printing or dip pen lithography are used to create tracks of streptavidin on the passivated surfaces to serve as anchoring points for the biotinylated peptide arginine-glycine-aspartic acid (RGD) as the adhesive cue. A microfluidic device is placed onto the modified surface and used to create the gradient of adhesive cues (100% RGD to 0% RGD) on the streptavidin tracks. Finally, the same microfluidic device is used to create a gradient of a chemoattractant such as fetal bovine serum (FBS), as the soluble cue in the opposite direction of the gradient of adhesive cues.

A method for the assembly of adhesive and soluble gradients in a microscopy chamber for live cell migration studies is described. The engineered environment combines antifouling surfaces and adhesive tracks with solution gradients and therefore allows one to determine the relative importance of guidance cues.

Cells can sense and migrate towards higher concentrations of adhesive cues such as the glycoproteins of the extracellular matrix and soluble cues such as ...

A Novel Method for Localizing Reporter Fluorescent Beads Near the Cell Culture Surface for Traction Force Microscopy

PA gels have long been used as a platform to study cell traction forces due to ease of fabrication and the ability to tune their elastic properties. When the substrate is coated with an extracellular matrix protein, cells adhere to the gel and apply forces, causing the gel to deform. The deformation depends on the cell traction and the elastic properties of the gel. If the deformation field of the surface is known, surface traction can be calculated using elasticity theory. Gel deformation is commonly measured by embedding fluorescent marker beads uniformly into the gel. The probes displace as the gel deforms. The probes near the surface of the gel are tracked. The displacements reported by these probes are considered as surface displacements. Their depths from the surface are ignored. This assumption introduces error in traction force evaluations. For precise measurement of cell forces, it is critical for the location of the beads to be known. We have developed a technique that utilizes simple chemistry to confine fluorescent marker beads, 0.1 and 1 &#181;m in diameter, in PA gels, within 1.6 &#956;m of the surface. We coat a coverslip with poly-D-lysine (PDL) and fluorescent beads. PA gel solution is then sandwiched between the coverslip and an adherent surface. The fluorescent beads transfer to the gel solution during curing. After polymerization, the PA gel contains fluorescent beads on a plane close to the gel surface.

Traditional techniques for fabricating polyacrylamide (PA) gels containing fluorescent probes involve sandwiching a gel between an adherent surface and a glass slide. Here, we show that coating this slide with poly-D-lysine (PDL) and fluorescent probes localizes the probes to within 1.6 &#181;m from the gel surface.

PA gels have long been used as a platform to study cell traction forces due to ease of fabrication and the ability to tune their elastic properties. When ...

Photoactivated Localization Microscopy with Bimolecular Fluorescence Complementation (BiFC-PALM)

Protein-protein interactions (PPIs) are key molecular events to biology. However, it remains a challenge to visualize PPIs with sufficient resolution and sensitivity in cells because the resolution of conventional light microscopy is diffraction-limited to ~250 nm. By combining bimolecular fluorescence complementation (BiFC) with photoactivated localization microscopy (PALM), PPIs can be visualized in cells with single molecule sensitivity and nanometer spatial resolution. BiFC is a commonly used technique for visualizing PPIs with fluorescence contrast, which involves splitting of a fluorescent protein into two non-fluorescent fragments. PALM is a recent superresolution microscopy technique for imaging biological samples at the nanometer and single molecule scales, which uses phototransformable fluorescent probes such as photoactivatable fluorescent proteins (PA-FPs). BiFC-PALM was demonstrated by splitting PAmCherry1, a PA-FP compatible with PALM, for its monomeric nature, good single molecule brightness, high contrast ratio, and utility for stoichiometry measurements. When split between amino acids 159 and 160, PAmCherry1 can be made into a BiFC probe that reconstitutes efficiently at 37 &#176;C with high specificity to PPIs and low non-specific reconstitution. Ras-Raf interaction is used as an example to show how BiFC-PALM helps to probe interactions at the nanometer scale and with single molecule resolution. Their diffusion can also be tracked in live cells using single molecule tracking (smt-) PALM. In this protocol, factors to consider when designing the fusion proteins for BiFC-PALM are discussed, sample preparation, image acquisition, and data analysis steps are explained, and a few exemplary results are showcased. Providing high spatial resolution, specificity, and sensitivity, BiFC-PALM is a useful tool for studying PPIs in intact biological samples.

Photoactivated Localization Microscopy with Bimolecular Fluorescence Complementation BiFC-PALM

Protein-protein interactions are visualized in cells with nanometer spatial resolution by combining bimolecular fluorescence complementation (BiFC) with photoactivated localization microscopy (PALM). Described here is the use of BiFC-PALM for imaging Ras-Raf interactions in U2OS cells for visualizing the nanoscale clustering and diffusion of individual Ras-Raf complexes.

Protein-protein interactions (PPIs) are key molecular events to biology. However, it remains a challenge to visualize PPIs with sufficient resolution and ...

Multicolor Fluorescence Detection for Droplet Microfluidics Using Optical Fibers

Fluorescence assays are the most common readouts used in droplet microfluidics due to their bright signals and fast time response. Applications such as multiplex assays, enzyme evolution, and molecular biology enhanced cell sorting require the detection of two or more colors of fluorescence. Standard multicolor detection systems that couple free space lasers to epifluorescence microscopes are bulky, expensive, and difficult to maintain. In this paper, we describe a scheme to perform multicolor detection by exciting discrete regions of a microfluidic channel with lasers coupled to optical fibers. Emitted light is collected by an optical fiber coupled to a single photodetector. Because the excitation occurs at different spatial locations, the identity of emitted light can be encoded as a temporal shift, eliminating the need for more complicated light filtering schemes. The system has been used to detect droplet populations containing four unique combinations of dyes and to detect sub-nanomolar concentrations of fluorescein.

Multicolor fluorescence detection in droplet microfluidics typically involves bulky and complex epifluorescence microscope-based detection systems. Here we describe a compact and modular multicolor detection scheme that utilizes an array of optical fibers to temporally encode multicolor data collected by a single photodetector.

Fluorescence assays are the most common readouts used in droplet microfluidics due to their bright signals and fast time response. Applications such as ...

A High-throughput Cell Microarray Platform for Correlative Analysis of Cell Differentiation and Traction Forces

Microfabricated cellular microarrays, which consist of contact-printed combinations of biomolecules on an elastic hydrogel surface, provide a tightly controlled, high-throughput engineered system for measuring the impact of arrayed biochemical signals on cell differentiation. Recent efforts using cell microarrays have demonstrated their utility for combinatorial studies in which many microenvironmental factors are presented in parallel. However, these efforts have focused primarily on investigating the effects of biochemical cues on cell responses. Here, we present a cell microarray platform with tunable material properties for evaluating both cell differentiation by immunofluorescence and biomechanical cell&#8211;substrate interactions by traction force microscopy. To do so, we have developed two different formats utilizing polyacrylamide hydrogels of varying Young&#39;s modulus fabricated on either microscope slides or glass-bottom Petri dishes. We provide best practices and troubleshooting for the fabrication of microarrays on these hydrogel substrates, the subsequent cell culture on microarrays, and the acquisition of data. This platform is well-suited for use in investigations of biological processes for which both biochemical (e.g., extracellular matrix composition) and biophysical (e.g., substrate stiffness) cues may play significant, intersecting roles.

Cell differentiation is regulated by a host of microenvironmental factors, including both matrix composition and substrate material properties. We describe here a technique utilizing cell microarrays in conjunction with traction force microscopy to evaluate both cell differentiation and biomechanical cell&#8211;substrate interactions as a function of microenvironmental context.

Microfabricated cellular microarrays, which consist of contact-printed combinations of biomolecules on an elastic hydrogel surface, provide a tightly ...

Fabrication and Validation of an Organ-on-chip System with Integrated Electrodes to Directly Quantify Transendothelial Electrical Resistance

Organs-on-chips, in vitro models involving the culture of (human) tissues inside microfluidic devices, are rapidly emerging and promise to provide useful research tools for studying human health and disease. To characterize the barrier function of cell layers cultured inside organ-on-chip devices, often transendothelial or transepithelial electrical resistance (TEER) is measured. To this end, electrodes are usually integrated into the chip by micromachining methods to provide more stable measurements than is achieved with manual insertion of electrodes into the inlets of the chip. However, these electrodes frequently hamper visual inspection of the studied cell layer or require expensive cleanroom processes for fabrication. To overcome these limitations, the device described here contains four easily integrated electrodes that are placed and fixed outside of the culture area, making visual inspection possible. Using these four electrodes the resistance of six measurement paths can be quantified, from which the TEER can be directly isolated, independent of the resistance of culture medium-filled microchannels. The blood-brain barrier was replicated in this device and its TEER was monitored to show the device applicability. This chip, the integrated electrodes and the TEER determination method are generally applicable in organs-on-chips, both to mimic other organs or to be incorporated into existing organ-on-chip systems.

This publication describes the fabrication of an organ-on-chip device with integrated electrodes for direct quantification of transendothelial electrical resistance (TEER). For validation, the blood-brain barrier was mimicked inside this microfluidic device and its barrier function was monitored. The presented methods for electrode integration and direct TEER quantification are generally applicable.

Organs-on-chips, in vitro models involving the culture of (human) tissues inside microfluidic devices, are rapidly emerging and promise to provide useful ...

Interfacing Microfluidics with Microelectrode Arrays for Studying Neuronal Communication and Axonal Signal Propagation

Microelectrode arrays (MEAs) are widely used to study neuronal function in vitro. These devices allow concurrent non-invasive recording/stimulation of electrophysiological activity for long periods. However, the property of sensing signals from all sources around every microelectrode can become unfavorable when trying to understand communication and signal propagation in neuronal circuits. In a neuronal network, several neurons can be simultaneously activated and can generate overlapping action potentials, making it difficult to discriminate and track signal propagation. Considering this limitation, we have established an in vitro setup focused on assessing electrophysiological communication, which is able to isolate and amplify axonal signals with high spatial and temporal resolution. By interfacing microfluidic devices and MEAs, we are able to compartmentalize neuronal cultures with a well-controlled alignment of the axons and microelectrodes. This setup allows recordings of spike propagation with a high signal-to-noise ratio over the course of several weeks. Combined with specialized data analysis algorithms, it provides detailed quantification of several communication related properties such as propagation velocity, conduction failure, firing rate, anterograde spikes, and coding mechanisms.
This protocol demonstrates how to create a compartmentalized neuronal culture setup over substrate-integrated MEAs, how to culture neurons in this setup, and how to successfully record, analyze and interpret the results from such experiments. Here, we show how the established setup simplifies the understanding of neuronal communication and axonal signal propagation. These platforms pave the way for new in vitro models with engineered and controllable neuronal network topographies. They can be used in the context of homogeneous neuronal cultures, or with co-culture configurations where, for example, communication between sensory neurons and other cell types is monitored and assessed. This setup provides very interesting conditions to study, for example, neurodevelopment, neuronal circuits, information coding, neurodegeneration and neuroregeneration approaches.

This protocol aims to demonstrate how to combine in vitro microelectrode arrays with microfluidic devices for studying action potential transmission in neuronal cultures. Data analysis, namely the detection and characterization of propagating action potentials, is performed using a new advanced, yet user-friendly and freely available, computational tool.

Microelectrode arrays (MEAs) are widely used to study neuronal function in vitro. These devices allow concurrent non-invasive recording/stimulation of ...

High Throughput Traction Force Microscopy Using PDMS Reveals Dose-Dependent Effects of Transforming Growth Factor-&#946; on the Epithelial-to-Mesenchymal Transition

Cellular contractility is essential in diverse aspects of biology, driving processes that range from motility and division, to tissue contraction and mechanical stability, and represents a core element of multi-cellular animal life. In adherent cells, acto-myosin contraction is seen in traction forces that cells exert on their substrate. Dysregulation of cellular contractility appears in a myriad of pathologies, making contractility a promising target in diverse diagnostic approaches using biophysics as a metric. Moreover, novel therapeutic strategies can be based on correcting the apparent malfunction of cell contractility. These applications,&#160;however, require direct quantification of these forces.
We have developed silicone elastomer-based traction force microscopy (TFM) in a parallelized multi-well format. Our use of a silicone rubber, specifically polydimethylsiloxane (PDMS), rather than the commonly employed hydrogel polyacrylamide (PAA) enables us to make robust and inert substrates with indefinite shelf-lives requiring no specialized storage conditions. Unlike pillar-PDMS based approaches that have a modulus in the GPa range, the PDMS used here is very compliant, ranging from approximately 0.4 kPa to 100 kPa. We create a high-throughput platform for TFM by partitioning these large monolithic substrates spatially into biochemically independent wells, creating a multi-well platform for traction force screening that is compatible with existing multi-well systems.
In this manuscript, we use this multi-well traction force system to examine the Epithelial to Mesenchymal Transition (EMT); we induce EMT in NMuMG cells by exposing them to TGF-&#946;, and to quantify the biophysical changes during EMT. We measure the contractility as a function of concentration and duration of TGF-&#946; exposure. Our findings here demonstrate the utility of parallelized TFM in the context of disease biophysics.

We present a high throughput traction force assay fabricated with silicone rubber (PDMS). This novel assay is suitable for studying physical changes in cell contractility during various biological and biomedical processes and diseases. We demonstrate this method's utility by measuring a TGF-&#946; dependent increase in contractility during the epithelial-to-mesenchymal transition.

Cellular contractility is essential in diverse aspects of biology, driving processes that range from motility and division, to tissue contraction and ...

Fabrication and Implementation of a Reference-Free Traction Force Microscopy Platform

Quantifying cell-induced material deformation provides useful information concerning how cells sense and respond to the physical properties of their microenvironment. While many approaches exist for measuring cell-induced material strain, here we provide a methodology for monitoring strain with sub-micron resolution in a reference-free manner. Using a two-photon activated photolithographic patterning process, we demonstrate how to generate mechanically and bio-actively tunable synthetic substrates containing embedded arrays of fluorescent fiducial markers to easily measure three-dimensional (3D) material deformation profiles in response to surface tractions. Using these substrates, cell tension profiles can be mapped using a single 3D image stack of a cell of interest. Our goal with this methodology is to make traction force microscopy a more accessible and easier to implement tool for researchers studying cellular mechanotransduction processes, especially newcomers to the field.

This protocol provides instructions for implementing multiphoton lithography to fabricate three-dimensional arrays of fluorescent fiducial markers embedded in poly(ethylene glycol)-based hydrogels for use as reference-free, traction force microscopy platforms. Using these instructions, measurement of 3D material strain and calculation of cellular tractions is simplified to promote high-throughput traction force measurements.

Quantifying cell-induced material deformation provides useful information concerning how cells sense and respond to the physical properties of their ...