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 ...

<ol>
	<li>Reig, G., Pulgar, E., Concha, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+migration:+from+tissue+culture+to+embryos.">Cell migration: from tissue culture to embryos.</a> Development. 141 (10), 1999-2013 (2014).</li><li>Luster, A. D., Alon, R., von Andrian, U. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune+cell+migration+in+inflammation:+present+and+future+therapeutic+targets.">Immune cell migration in inflammation: present and future therapeutic targets.</a> Nature Immunology. 6 (12), 1182-1190 (2005).</li><li>Liang, C. C., Park, A. Y., Guan, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+scratch+assay:+a+convenient+and+inexpensive+method+for+analysis+of+cell+migration+in+vitro.">In vitro scratch assay: a convenient and inexpensive method for analysis of cell migration in vitro.</a> Nature Protocols. 2 (2), 329-333 (2007).</li><li>Friedl, P., Gilmour, D. <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+migration+in+morphogenesis,+regeneration+and+cancer.">Collective cell migration in morphogenesis, regeneration and cancer.</a> Nature Reviews Molecular Cell Biology. 10 (7), 445-457 (2009).</li><li>Mayor, R., Etienne-Manneville, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+front+and+rear+of+collective+cell+migration.">The front and rear of collective cell migration.</a> Nature Reviews Molecular Cell Biology. 17 (2), 97-109 (2016).</li><li>DuFort, C. C., Paszek, M. J., Weaver, V. 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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 border="0" cellpadding="0" cellspacing="0" style="width:1461px;" width="1460">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="22">
			<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 ...

Collective cell migration is often guided by the gradient of signaling molecules. In this video, we demonstrate how to quantify cellular forces during collective migration guided by biochemical gradient. So, we integrate the microfluidic chip with traction microscopy, then we use the microfluidic chip to generate biochemical gradient, and we use the traction microscopy to measure cellular force.Dr.Hwanseok Jang, a postgrad in my laboratory, will demonstrate the procedure. Prepare the PDMS solution by mixing the base elastomer and curing agent at a ratio of ten to one. Place 15 milliliters of the base elastomer in a 50 milliliter conical tube, and add 1.5 milliliters of curing agent.Prepare two of these tubes. Vortex the PDMS mixture for five minutes before centrifuging at 196 time g for one minute to remove bubbles. To fabricate the PDMS stencil, pour approximately one milliliter 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.Place the wafer on a flat surface for over 30 minutes at room temperature, then cure the PDMS in a dry oven at 80 degrees Celsius for over two hours. Carefully peel off the PDMS from SU-8 mold, trim the thin PDMS membrane using a 14 millimeter hollow punch. Remove the dust on the surface of the PDMS pieces using sticky tape before autoclaving the PDMS stencils.To fabricate PDMS microchannel, pour approximately 30 milliliters of PDMS mixture over the SU-8 mold. Degas for 30 minutes in a vacuum chamber, and then cure the PDMS in a dry oven at 80 degrees Celsius for over two hours. Carefully peel off the PDMS from SU-8 mold, and cut the PDMS to a size of 24 millimeters by 24 millimeters.In each PDMS block, create one outlet and three inlets using a one millimeter biopsy punch. Manufacture and silanize glass slides as described in the text protocol. Cover the edged surface of the bottom glass with 100 microliters of the bind-silane solution.After leaving the glass at room temperature for one hour, rinse the glass three times with deionized water. Then let the glass dry at ambient air temperature or by blowing compressed air. Prepare the polyacrylamide gel solution as described in the text protocol.Then transfer for 10 microliters of mixed gel solution onto the rectangular microwell and place a circular cover slip on top. Fix the assembly of custom glass, gel solution, and cover slip on the cover of a six-well plate with sticky tape and flip it. Then centrifuge for 10 minutes at 96 times g to bring fluorescent particles to the top layer of the polyacrylamide gel.Remove the assembly from the centrifuge and place it on a flat surface with the cover slip facing down. After 30 minutes, flip the assembly and place it in a 35 millimeter Petri dish. Fill the dish with two milliliters of deionized water.Using forceps, gently remove the cover slip by sliding it to one side. To code collagen on the polyacrylamide gel, dissolve one milligram per milliliter sulfo-SANPAH in a warm 50 millimolar HEPES buffer. Drop 200 microliters of the solution onto the gel surface and activate by UV light for 10 minutes.Following UV activation, rinse the gel two times with 0.1 molar HEPES buffer, and then once with PBS. Code the polyacrylamide gel with collagen solution at four degrees Celsius overnight. On the following day, wash the gel three times with PBS.Immerse the autoclaved PDMS stencil in F-127 solution. Keep it in a 37-degree Celsius incubator for one hour. Wash the PDMS stencil with PBS three times, and remove liquid from both the PDMS stencil and polyacrylamide gel.Then place the PDMS stencil on the polyacrylamide gel, and add PBS to the stencil. Remove bubbles in the holes of the PDMS stencil by pipetting gently. After removing bubbles, clear the PBS from the surface of the PDMS stencil.Now add 200 microliters of the cell solution onto PDMS stencil and place the gel in an incubator for one hour so that the cells attach to the polyacrylamide gel. Following incubation, gently wash off the cell solution with cell culture media and add more cell culture media. Remove the PDMS stencil, check the formation of cell islands under a microscope.Next, treat the surface of the PDMS microchannel with oxygen plasma for 30 seconds. After removing any fluid on the polyacrylamide gel-filled bottom glass, place the PDMS microchannel on top of the bottom glass, and put the assembly on the custom glass holder. Fill the microchannel with cell culture medium.To prepare the inlet tubing of the integrated microfluidic system, connect a trimmed needle and a 30-centimeter mini volume line with a three-way stopcock. Prepare three of these arrangements. For outlet tubing, connect a trimmed needle and a 75-centimeter mini volume line with a three-way stopcock.Fill the tubing lines with the medium that has been preheated for one hour. Prepare reservoirs by removing plungers from syringes and connecting inlet tubing lines. Plug the needle connectors of each tubing line into the three inlets and one outlet of the microfluidic device.Fill each reservoir with three milliliters of fresh medium or conditioned medium. For the gradient test, fill the left inlet reservoir with 20 nanograms per milliliter hepatocyte growth factor, or HGF, in the cell culture medium. For visualization of the concentration gradient, add 200 micrograms per milliliter of fluorescent dye to the left inlet reservoir.Connect the outlet tubing line to a syringe pump. Place the integrated microfluidic system on the stage of a conventional epifluorescent microscope. Take images every 10 minutes for up to 24 hours using an automated microscope housed in an incubator.At each time point, take a set of images using a 4X objective lens in three different channels, including a phase image to visualize cell migration, a green fluorescent image to visualize fluorescent beads embedded in the gel, and a red fluorescent image to visualize the concentration gradient of a chemical. After taking time-lapse images, infuse 0.25%Trypsin-EDTA solution into microchannels to detach cells from the polyacrylamide gel. Upon completely removing cells from the gel, take a green fluorescent image to be used as a reference image for traction microscopy.Proceed to data analysis as described in the text protocol. To measure contractile force and intercellular stress, traction microscopy and monolayer stress microscopy were combined with the microfluidic channel. The maps were plotted in radio coordination with outward traction using warm color and inward traction using cold color.At time zero, all islands showed similar traction distributions with strong inward traction on the edge and fluctuation within the island. After 10 hours of applying the HGF gradient, while the degree of island expansion was different in each column, the traction distributions were largely similar to time zero. The average traction did not change for 10 hours.When calculating the monolayer stress, however, each column showed different trends. Where HGF concentration was low, the average tension within islands was maintained around 200 pascals throughout a 10-hour period. Where HGF concentration was high, the average tension within islands gradually decreased from 230 pascals to 100 pascals over 10 hours, as previously shown.Where the HGF concentration was high on the left half and low on the right half of the island, the average tension was maintained around 150 pascals. Filling the microfluidic channel must be gentle. It is important to remove bubbles trapped in the channel while simultaneously not disrupting micro parent cell islands.By using this integrated system, one can investigate it how cell collective respond mechanically to various chemical gradients, such as chemo attractants or growth factors. This platform will help us further explore the mechanics of collective cell migration under chemical gradients, which is the key to understand regeneration, cancer metastasis, and development.

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

Research

JoVE Journal

Bioengineering

6.9K visualizações.  Korea University. A migração celular coletiva é frequentemente guiada pelo gradiente das moléculas de sinalização. Neste vídeo, demonstramos como quantificar as forças celulares durante a migração coletiva guiada pelo gradiente bioquímico. Então, integramos o chip microfluido com microscopia de tração, então usamos o chip microfluido para gerar gradiente bioquímico, e usamos a microscopia de tração para medir a força celular.Dr.Hwanseok Jang, um pós-graduado no meu laboratório, vai ...