Traction Microscopy Integrated with Microfluidics for Chemotactic Collective Migration

Summary

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.

Abstract

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.

Introduction

Cellular migration in biological systems is a fundamental phenomenon involved in tissue formation, the immune response, and wound healing^1,2,3. 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 migration^4,5. For cells to move collectively, sensing of the microenvironment is essential⁶. For instance, cells perceive physicochemical stimuli and respond by ....

Protocol

NOTE: Lithography of SU-8 molds for stencils (thickness = 250 μm) and microchannel parts (thickness = 150 μm), glass etching (depth = 100 μm), and cast fabrication were outsourced by sending designs using computer-aided design software to manufacturers.

1. Fabrication of polydimethylsiloxane (PDMS) stencil and microchannel

1. Design the micropattern of stencil and microchannel.
2. Fabricate or outsource SU-8 molds (thickness of ~250 μm for stencils and ~150 μm for microchannels) on silicon wafers (4" diameter).
3. Prepare PDMS mixture by mixing the base elastomer and curing agent at a....

Results

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 μm) were created. After cells attached to PA gels, the PDMS stencil was removed to initiate col.......

Discussion

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 factors^4,23. 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

Materials

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