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.

<table><tbody><tr><td>0.25% trypsin-EDTA (1X)</td><td>Gibco</td><td>25200-056</td><td></td></tr><tr><td>1 M HEPES buffer solution</td><td>Gibco</td><td>15630-056</td><td></td></tr><tr><td>1 mm Biopsy punch</td><td>Integra Miltex</td><td>33-31AA-P/25</td><td></td></tr><tr><td>100 mm petri dishes</td><td>SPL</td><td>10100</td><td>100 mm diameter, 15 mm height</td></tr><tr><td>14 mm hollow punch</td><td>ILJIN</td><td>124-0571</td><td></td></tr><tr><td>18 mm &amp;Oslash; Coverslip</td><td>Marienfeld-Superior</td><td>111580</td><td>Circular 18 mm, thickness No. 1 (0.13 to 0.16 mm)</td></tr><tr><td>2% bis-acrylamide solution</td><td>Bio-Rad</td><td>1610142</td><td>Wear protective gloves, clothing, and eye protection.</td></tr><tr><td>3-(Trimethoxysilyl)propyl methacrylate (TMSPMA)</td><td>Sigma-Aldrich</td><td>440159-500ML</td><td></td></tr><tr><td>3-way stopcock</td><td>Hyupsung</td><td>HS-T-61N</td><td>CAUTION: do not use if previously opened. do not resterlize or resuse</td></tr><tr><td>30 cm minimum volume line (for pediatric)</td><td>Hyupsung</td><td>HS-MV-30</td><td>CAUTION: do not use if previously opened. do not resterlize or resuse</td></tr><tr><td>35 mm cell culture dish</td><td>Corning</td><td>430165</td><td></td></tr><tr><td>40% Acrylamide Solution</td><td>Bio-Rad</td><td>1610140</td><td>Wear protective gloves, clothing, and eye protection.</td></tr><tr><td>75 cm minimum volume line (for pediatric)</td><td>Hyupsung</td><td>HS-MV-75</td><td>CAUTION: do not use if previously opened. do not resterlize or resuse</td></tr><tr><td>acetic acid</td><td>J.T. Baker</td><td>JT9508-03</td><td></td></tr><tr><td>Ammonium persulfate (APS)</td><td>Bio-Rad</td><td>1610700</td><td></td></tr><tr><td>Antibiotic-Antimycotic</td><td>Gibco</td><td>15240-062</td><td></td></tr><tr><td>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 &amp;mu;m)</td></tr><tr><td>Collagen typeI, Rat tail</td><td>Corning</td><td>354236</td><td></td></tr><tr><td>Custom glass holder</td><td>Han-Gug Mechatronics</td><td>custom-made</td><td></td></tr><tr><td>Dulbecco&#039;s Modified Eagle&#039;s Medium (DMEM)</td><td>Welgene</td><td>LM 001-11</td><td></td></tr><tr><td>Dulbecco&#039;s Phosphate Buffered Saline (PBS)</td><td>Biowest</td><td>L0615-500</td><td>w/o Magnesium, Calcium</td></tr><tr><td>Fetal bovine serum (FBS)</td><td>Gibco</td><td>26140-179</td><td></td></tr><tr><td>FluoSpheres amine-modified microspheres</td><td>Invitrogen</td><td>F8764</td><td>0.2 &amp;micro;m, yellow-green fluorescent(505/515)</td></tr><tr><td>Hepatocyte Growth Factor (HGF)</td><td>Sigma-Aldrich</td><td>H1404-5UG</td><td>recombinant, human</td></tr><tr><td>JuLI stage live cell imaging system</td><td>NanoEnTek In</td><td></td><td>Automated X-Y-Z stage and fluorsent imaging Incubator-compatible (37 &amp;deg;C and 5% CO2)</td></tr><tr><td>Madin-Darby Canine Kidney (MDCK) cell</td><td></td><td></td><td>type II</td></tr><tr><td>Oxygen plasma system</td><td>Femto Science</td><td>CUTE-MPR</td><td></td></tr><tr><td>Pluronic F-127</td><td>Sigma-Aldrich</td><td>P2443-250G</td><td></td></tr><tr><td>Rhodamine B isothiocyanate&amp;ndash;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><td>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><td>Sticky tape</td><td>3M/Scotch</td><td>810D</td><td>33 m x 19 mm</td></tr><tr><td>SU-8 master molds</td><td>MicroFIT</td><td></td><td>4&amp;rdquo; diameter, custom-made</td></tr><tr><td>sulfosuccinimidyl 6-(4&amp;rsquo;-azido-2&amp;rsquo;-nitrophenylamino)hexanoate (Sulfo-SANPAH)</td><td>Thermo Scientific</td><td>22589</td><td>Store at -20&amp;deg;C. Store protected from moisture and light.</td></tr><tr><td>Sylgard 184 Elastomer Kit</td><td>Dow Corning</td><td></td><td>PDMS</td></tr><tr><td>Syringe pump</td><td>Chemyx Inc.</td><td>model fusion 720</td><td>withdraw fluid</td></tr><tr><td>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><td>tetramethylethylenediamine (TEMED)</td><td>Bio-Rad</td><td>1610800</td><td>Wear protective gloves, clothing, and eye protection.</td></tr><tr><td>Ultraviolet (UV) lamp</td><td>UVP LLC</td><td>95-0248-02</td><td>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

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

7.0K 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

Chemotactic Response of Marine Micro-Organisms to Micro-Scale Nutrient Layers

The degree to which planktonic microbes can exploit microscale resource patches will have considerable implications for oceanic trophodynamics and biogeochemical flux. However, to take advantage of nutrient patches in the ocean, swimming microbes must overcome the influences of physical forces including molecular diffusion and turbulent shear, which will limit the availability of patches and the ability of bacteria to locate them. Until recently, methodological limitations have precluded direct examinations of microbial behaviour within patchy habitats and realistic small-scale flow conditions. Hence, much of our current knowledge regarding microbial behaviour in the ocean has been procured from theoretical predictions. To obtain new information on microbial foraging behaviour in the ocean we have applied soft lithographic fabrication techniques to develop 2 microfluidic devices, which we have used to create (i) microscale nutrient patches with dimensions and diffusive characteristics relevant to oceanic processes and (ii) microscale vortices, with shear rates corresponding to those expected in the ocean. These microfluidic devices have permitted a first direct examination of microbial swimming and chemotactic behaviour within a heterogeneous and dynamic seascape. The combined use of epifluorescence and phase contrast microscopy allow direct examinations of the physical dimensions and diffusive characteristics of nutrient patches, while observing the population-level aggregative response, in addition to the swimming behaviour of individual microbes. These experiments have revealed that some species of phytoplankton, heterotrophic bacteria and phagotrophic protists are adept at locating and exploiting diffusing microscale resource patches within very short time frames. We have also shown that up to moderate shear rates, marine bacteria are able to  fight the flow  and swim through their environment at their own accord. However, beyond a threshold high shear level, bacteria are aligned in the shear flow and are less capable of swimming without disturbance from the flow. Microfluidics represents a novel and inexpensive approach for studying aquatic microbial ecology, and due to its suitability for accurately creating realistic flow fields and substrate gradients at the microscale, is ideally applicable to examinations of microbial behaviour at the smallest scales of interaction.  We therefore suggest that microfluidics represents a valuable tool for obtaining a better understanding of the ecology of microorganisms in the ocean.

The fabrication of microfluidic channels and their implementation in experiments for studying the chemotactic foraging behaviour of marine microbes within a patchy nutrient seascape and the swimming behaviour of bacteria within shear flow are described.

The degree to which planktonic microbes can exploit microscale resource patches will have considerable implications for oceanic trophodynamics and ...

Biology

Studies of Bacterial Chemotaxis Using Microfluidics - Interview

My name is Roman Stocker. I'm a second year assistant professor in the Ralph Parsons Laboratory, which is part of the Department of Civil and Environmental Engineering here at MIT. I'm originally from Italy and I studied fluid mechanics.My background is therefore more in the physical sciences, but in the past two years, three years actually, I've moved more into environmental and biological topics. Two years ago, I've started developing microfluidics to look at problems in microbial ecology and using these micro devices, we are able to develop typical ecological questions for things at the scales of microns. Our work is primarily concerned with al organisms, those that can swim around in the ocean and track their sources of food.In particular, bacteria have developed very clever ways of swimming, and I have a small demonstration here. The bacterial head is pretty much an ellipsoid. You imagine this being about two microns in size.At the back of the bacterium, there is one or more flagella. These flagella are helical structures, which the bacterium can rot rotate. That rotation pro produces trust that allows the bacterium to actually swim forward.There is different modes in which bacteria actually do that, and there are different strategies of swimming and it is part of what our research is concerned with. The reason bacteria swim in the ocean is to be able to better exploit sources of nutrients, and that is part of a paradigm shift which has occurred over the last 15 or 20 years. Before that, people used to think that the ocean was a homogeneous soup of nutrients at scales of meters and up to tens of meters, and in that picture of the ocean, these small scale organisms like bacteria were taught to play no substantial role and they were really just passively vectored by the oceanic occurrence.But 10 or 15 years ago, people realized that that is really not what is going on in the ocean and that in fact these small scale organisms are among the most important players in determining the ocean health and therefore even things like our global climate. This is so because of two reasons. The first reason is that they occur everywhere in the ocean and their cumulative biomass by far exceeds that of all other marine organisms.The second reason, and maybe the most fundamental one, is that these small scale organisms by utilizing nutrient sources and in particular carbon, can recycle the nutrients for other components of the food web and therefore substantially intensifying other aspects and other parts of the trophic chain in the ocean. In all of this, our work focuses primarily on interaction between swimming organisms and nutrient patches. Bacteria not only are able to swim, but they also able to sense detect nutrient gradients.That is, they're able to tell when life is getting better and swim up gradients of nutrients. They do that in a process known as a chemotaxis.Properly. Chemo taxes is the ability to sense a chemical gradient and by swimming home in on it.This is really what we study in the lab and microfluidics is a tool that allows us to probe the chemotactic and swimming abilities of bacteria and therefore extrapolate and scale up their role to determine how important they are in the determining the health of the ocean. In the Past year and a half, we've set up micro devices that allow us to expose bacteria to different kind of conditions that you typically find in the ocean. As I said earlier, typical nutrient patches, but also for example, turbulence.The what we have found so far is that marine bacteria seem to have adapted very efficiently to exploit these nutrient patches and to be able to counteract by swimming a large variety of conditions of turbulence conditions in the ocean that would make them very well suited to actually survive in an ocean, which is typically a desert of nutrients from their point of view and in which nutrients occur only in scattered and very rare patches in a three-dimensional water column, and at the same time be able to swim, to fight turbulence and therefore still be able to track their nutrient foods even when the water around them is moving violently. The Earth system initiative is a very interesting initiative and one that brings together a variety of people from different disciplines, all interested in understanding better how our planet actually works. In short, the earth system initiative's goal is to try to do the same thing for the planet as we are already doing and have been doing for a long time.For the human body that is understanding its health, understanding the health of the planet these days is particularly important because of concerns of global warming, for example, and greenhouse gases and several of the efforts within the earth system initiative are geared towards understanding the health of the planet better. Our work fits into that by looking at one component of the carbon cycle in the ocean. We try to understand in particular how small scale organisms, as I mentioned earlier, swimming bacteria in particular can affect these large scale cycles, which has repercussions on the global carbon cycle in the ocean and therefore on the global carbon cycle worldwide, including the Atmosphere.All of our work So far in this field has been allowed and actually triggered by one key technological advance, and that is microfluidics. In the past 10 years, microfluidics has been developed into a really interesting and cool tool in particular in the Boston Cambridge area. The originator of the microfluidics technology in its current form is Professor George Whitesides at Harvard who introduced the process of soft lithography and the use of PDMS for micro devices fabrication.Since then, the application of microfluidics has grown exponentially in a whole range of fields. From chemical engineering to the health sciences, we have started applying it to microbial ecology and to address some of these small scale problems in in the ocean regarding the swimming of microorganisms. In a nutshell, it is difficult, if not impossible to study these processes directly in the ocean because of their small scales and because of the variability that the ocean necessarily presents, we are still not able to visualize bacteria which are about a micron in size, directly in situ in the ocean, and at the same time, people have tried to model these processes, for example, numerically or theoretically, but because this is biology, you necessarily have to rely on assumptions.For example, you will have to make assumptions about the swimming strategy of a bacteria or the chemo taxi strategy of a bacterium, and therefore real experiments are needed to make progress in this field. Microfluidics allows us to take small regions, small parts of the ocean into the lab, and at the same time expose bacteria and microorganisms to environmental conditions that are realistic, both in terms of nutrients and in terms of flows. At the same time, by using video microscopy and cell tracking, we can study the behavior of individual bacteria in a very carefully controlled manner that allows us to make progress in these questions in a way that would not have been possible about 10 years ago, and all of that is due to technological advances, specifically micro Verdicts.There are two main challenges I see lying ahead of us in this field. The first one, as I mentioned earlier, is to be able at some point to take these questions to the ocean. The reason that one ultimately needs to go to the ocean is to capture all the variability that is inherent in these processes.For example, we still have no idea of what really leaks out of a settling particle, what kind of nutrients are present, what kind of chemical compounds do bacteria chemo tax towards in the lab. We can only study the simplest of these conditions, but what really occurs in the ocean is a collection of complicated and very variable processes that we need to ultimately explore direct directly in C two. This is a big challenge from the point of view of sampling from a point of view of imaging and from the point of view of doing chemical and biological analysis on scales, which as I mentioned a few times are only in the order of a few micrometers and in terms of volumes of picoliters.The second challenge that I see more easier to tackle is that visualizing behavior in three dimensions. Everything we do currently in the lab is two dimensional. We use conventional microscopy to visualize trajectories and accumulation of microorganisms, but we have great hope in a technique known as digital holography for giving us in the timeframe of maybe three to five years the ability to track these things in three dimensions and therefore more fatefully and more realistically, realistically be able to capture the ecology In the next Five years.In addition to trying to go three dimensional in the sampling and perhaps trying to make the first, first naive steps possibly towards the sampling things in the ocean, we definitely want to link these studies with ongoing efforts to understand genomics of these organisms. There is a big push in particular as part of the earth system initiative in understanding the genetic composition and the variability in terms of genetics of these bacteria out there in the field. At the same time, genomics lacks the ability of directly linking the findings with the behavior of the organisms, and that is what we hope that microfluidics and these more behavioral, classical ecological studies will be able to bring to the table.We have a great setup and a great team of people looking at the genomics of marine bacteria, and by collaborating with them, we hope to be able to link these two fundamental questions to get a better understanding of what bacteria out there in the ocean and what their role is in determining the health of the ocean and health of the planet.

Evaluation of Cancer Stem Cell Migration Using Compartmentalizing Microfluidic Devices and Live Cell Imaging

In the last 40 years, the United States invested over 200 billion dollars on cancer research, resulting in only a 5% decrease in death rate. A major obstacle for improving patient outcomes is the poor understanding of mechanisms underlying cellular migration associated with aggressive cancer cell invasion, metastasis and therapeutic resistance1. Glioblastoma Multiforme (GBM), the most prevalent primary malignant adult brain tumor2, exemplifies this difficulty. Despite standard surgery, radiation and chemotherapies, patient median survival is only fifteen months, due to aggressive GBM infiltration into adjacent brain and rapid cancer recurrence2. The interactions of aberrant cell migratory mechanisms and the tumor microenvironment likely differentiate cancer from normal cells3. Therefore, improving therapeutic approaches for GBM require a better understanding of cancer cell migration mechanisms. Recent work suggests that a small subpopulation of cells within GBM, the brain tumor stem cell (BTSC), may be responsible for therapeutic resistance and recurrence. Mechanisms underlying BTSC migratory capacity are only starting to be characterized1,4.
Due to a limitation in visual inspection and geometrical manipulation, conventional migration assays5 are restricted to quantifying overall cell populations. In contrast, microfluidic devices permit single cell analysis because of compatibility with modern microscopy and control over micro-environment6-9.
We present a method for detailed characterization of BTSC migration using compartmentalizing microfluidic devices. These PDMS-made devices cast the tissue culture environment into three connected compartments: seeding chamber, receiving chamber and bridging microchannels. We tailored the device such that both chambers hold sufficient media to support viable BTSC for 4-5 days without media exchange. Highly mobile BTSCs initially introduced into the seeding chamber are isolated after migration though bridging microchannels to the parallel receiving chamber. This migration simulates cancer cellular spread through the interstitial spaces of the brain. The phase live images of cell morphology during migration are recorded over several days. Highly migratory BTSC can therefore be isolated, recultured, and analyzed further. 
Compartmentalizing microfluidics can be a versatile platform to study the migratory behavior of BTSCs and other cancer stem cells. By combining gradient generators, fluid handling, micro-electrodes and other microfluidic modules, these devices can also be used for drug screening and disease diagnosis6. Isolation of an aggressive subpopulation of migratory cells will enable studies of underlying molecular mechanisms.

A compartmentalizing microfluidic device for investigating cancer stem cell migration is described. This novel platform creates a viable cellular microenvironment and enables microscopic visualization of live cell locomotion. Highly motile cancer cells are isolated to study molecular mechanisms of aggressive infiltration, potentially leading to more effective future therapies.

In the last 40 years, the United States invested over 200 billion dollars on cancer research, resulting in only a 5% decrease in death rate. A major ...

Medicine

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 ...

Study of Cell Migration in Microfabricated Channels

The method described here allows the study of cell migration under confinement in one dimension. It is based on the use of microfabricated channels, which impose a polarized phenotype to cells by physical constraints. Once inside channels, cells have only two possibilities: move forward or backward. This simplified migration in which directionality is restricted facilitates the automatic tracking of cells and the extraction of quantitative parameters to describe cell movement. These parameters include cell velocity, changes in direction, and pauses during motion. Microchannels are also compatible with the use of fluorescent markers and are therefore suitable to study localization of intracellular organelles and structures during cell migration at high resolution. Finally, the surface of the channels can be functionalized with different substrates, allowing the control of the adhesive properties of the channels or the study of haptotaxis. In summary, the system here described is intended to analyze the migration of large cell numbers in conditions in which both the geometry and the biochemical nature of the environment are controlled, facilitating the normalization and reproducibility of independent experiments.

A quantitative method to study spontaneous migration of cells in a one-dimensional confined microenvironment is described. This method takes advantage of microfabricated channels and can be used to study migration of large number of cells under different conditions in single experiments.

The method described here allows the study of cell migration under confinement in one dimension. It is based on the use of microfabricated channels, which ...

In Situ Chemotaxis Assay to Examine Microbial Behavior in Aquatic Ecosystems

Microbial behaviors, such as motility and chemotaxis (the ability of a cell to alter its movement in response to a chemical gradient), are widespread across the bacterial and archaeal domains. Chemotaxis can result in substantial resource acquisition advantages in heterogeneous environments. It also plays a crucial role in symbiotic interactions, disease, and global processes, such as biogeochemical cycling. However, current techniques restrict chemotaxis research to the laboratory and are not easily applicable in the field. Presented here is a step-by-step protocol for the deployment of the in situ chemotaxis assay (ISCA), a device that enables robust interrogation of microbial chemotaxis directly in the natural environment. The ISCA is a microfluidic device consisting of a 20 well array, in which chemicals of interest can be loaded. Once deployed in aqueous environments, chemicals diffuse out of the wells, creating concentration gradients that microbes sense and respond to by swimming into the wells via chemotaxis. The well contents can then be sampled and used to (1) quantify strength of the chemotactic responses to specific compounds through flow cytometry, (2) isolate and culture responsive microorganisms, and (3) characterize the identity and genomic potential of the responding populations through molecular techniques. The ISCA is a flexible platform that can be deployed in any system with an aqueous phase, including marine, freshwater, and soil environments.

Presented here is the protocol for an in situ chemotaxis assay, a recently developed microfluidic device that enables studies of microbial behavior directly in the environment.

Microbial behaviors, such as motility and chemotaxis (the ability of a cell to alter its movement in response to a chemical gradient), are widespread ...

Environment

Planar Gradient Diffusion System to Investigate Chemotaxis in a 3D Collagen Matrix

The importance of cell migration can be seen through the development of human life. When cells migrate, they generate forces and transfer these forces to their surrounding area, leading to cell movement and migration. In order to understand the mechanisms that can alter and/or affect cell migration, one can study these forces. In theory, understanding the fundamental mechanisms and forces underlying cell migration holds the promise of effective approaches for treating diseases and promoting cellular transplantation. Unfortunately, modern chemotaxis chambers that have been developed are usually restricted to two dimensions (2D) and have complex diffusion gradients that make the experiment difficult to interpret. To this end, we have developed, and describe in this paper, a direct-viewing chamber for chemotaxis studies, which allows one to overcome modern chemotaxis chamber obstacles able to measure cell forces and specific concentration within the chamber in a 3D environment to study cell 3D migration. More compelling, this approach allows one to successfully model diffusion through 3D collagen matrices and calculate the coefficient of diffusion of a chemoattractant through multiple different concentrations of collagen, while keeping the system simple and user friendly for traction force microscopy (TFM) and digital volume correlation (DVC) analysis.

Cell migration is an important part of human development and life. In order to understand the mechanisms that can alter cell migration, we present a planar gradient diffusion system to investigate chemotaxis in a 3D collagen matrix, which allows one to overcome modern diffusion chamber limitations of existing assays.

The importance of cell migration can be seen through the development of human life. When cells migrate, they generate forces and transfer these forces to ...

A Microfluidic Device for Quantifying Bacterial Chemotaxis in Stable Concentration Gradients

Chemotaxis allows bacteria to approach sources of attractant chemicals or to avoid sources of repellent chemicals. Bacteria constantly monitor the concentration of specific chemoeffectors by comparing the current concentration to the concentration detected a few seconds earlier. This comparison determines the net direction of movement. Although multiple, competing gradients often coexist in nature, conventional approaches for investigating bacterial chemotaxis are suboptimal for quantifying migration in response to concentration gradients of attractants and repellents. Here, we describe the development of a microfluidic chemotaxis model for presenting precise and stable concentration gradients of chemoeffectors to bacteria and quantitatively investigating their response to the applied gradient. The device is versatile in that concentration gradients of any desired absolute concentration and gradient strength can be easily generated by diffusive mixing. The device is demonstrated using the response of Escherichia coli RP437 to gradients of amino acids and nickel ions.

This protocol describes the development of a microfluidic device for investigating bacterial chemotaxis in stable concentration gradients of chemoeffectors.

Chemotaxis allows bacteria to approach sources of attractant chemicals or to avoid sources of repellent chemicals. Bacteria constantly monitor the ...

Time-lapse Imaging of Mouse Macrophage Chemotaxis

Chemotaxis is receptor-mediated guidance of cells along a chemical gradient, whereas chemokinesis is the stimulation of random cell motility by a chemical. Chemokinesis and chemotaxis are fundamental for the mobilization and deployment of immune cells. For example, chemokines (chemotactic cytokines) can rapidly recruit circulating neutrophils and monocytes to extravascular sites of inflammation. Chemoattractant receptors belong to the large family of G protein-coupled receptors. How chemoattractant (i.e., ligand) gradients direct cell migration via G protein-coupled receptor signaling is not yet fully understood. In the field of immunology, neutrophils are popular model cells for studying chemotaxis in vitro. Here we describe a real-time two-dimensional (2D) chemotaxis assay tailored for mouse resident macrophages, which have traditionally been more difficult to study. Macrophages move at a slow pace of ~1 &#181;m/min on a 2D surface and are less well suited for point-source migration assays (e.g., migration towards the tip of a micropipette filled with chemoattractant) than neutrophils or Dictyostelium discoideum, which move an order of magnitude faster. Widely used Transwell assays are useful for studying the chemotactic activity of different substances, but do not provide information on cell morphology, velocity, or chemotactic navigation. Here we describe a time-lapse microscopy-based macrophage chemotaxis assay that allows quantification of cell velocity and chemotactic efficiency and provides a platform to delineate the transducers, signal pathways, and effectors of chemotaxis.

Here we describe methods using time-lapse, phase-contrast microscopy to image mouse resident peritoneal macrophages in a chemotactic complement C5a gradient. The protocols can be extended to other immune cells.

Chemotaxis is receptor-mediated guidance of cells along a chemical gradient, whereas chemokinesis is the stimulation of random cell motility by a ...

Immunology and Infection

A Customizable Chamber for Measuring Cell Migration

Cell migration is a vital part of immune responses, growth, and wound healing. Cell migration is a complex process that involves interactions between cells, the extracellular matrix, and soluble and non-soluble chemical factors (e.g., chemoattractants). Standard methods for measuring the migration of cells, such as the Boyden chamber assay, work by counting cells on either side of a divider. These techniques are easy to use; however, they offer little geometric modification for different applications. In contrast, microfluidic devices can be used to observe cell migration with customizable concentration gradients of soluble factors1,2. However, methods for making microfluidics based assays can be difficult to learn.
Here, we describe an easy method for creating cell culture chambers to measure cell migration in response to chemical concentration gradients. Our cell migration chamber method can create different linear concentration gradients in order to study cell migration for a variety of applications. This method is relatively easy to use and is typically performed by undergraduate students.
The microchannel chamber was created by placing an acrylic insert in the shape of the final microchannel chamber well into a Petri dish. After this, poly(dimethylsiloxane) (PDMS) was poured on top of the insert. The PDMS was allowed to harden and then the insert was removed. This allowed for the creation of wells in any desired shape or size. Cells may be subsequently added to the microchannel chamber, and soluble agents can be added to one of the wells by soaking an agarose block in the desired agent. The agarose block is added to one of the wells, and time-lapse images can be taken of the microchannel chamber in order to quantify cell migration. Variations to this method can be made for a given application, making this method highly customizable.

This protocol details a customizable method to measure cell migration in response to chemoattractants that may also be used to determine the diffusion rate of a drug out of a polymer matrix.

Cell migration is a vital part of immune responses, growth, and wound healing. Cell migration is a complex process that involves interactions between ...