Name: traction microscopy integrated with microfluidics for chemotactic collective migration
Uploaded: 2019-10-13T15:00:00
Description: Watch this Scientific Journal Video about Traction Microscopy Integrated with Microfluidics for Chemotactic Collective Migration at JoVE.com

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. 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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">
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	<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 ...

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