Sign In

A subscription to JoVE is required to view this content. Sign in or start your free trial.

In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

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 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 ....

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 .......

Representative 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 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

Acknowledgements

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).

....

Materials

NameCompanyCatalog NumberComments
0.25% trypsin-EDTA (1X)Gibco25200-056
1 M HEPES buffer solutionGibco15630-056
1 mm Biopsy punchIntegra Miltex33-31AA-P/25
100 mm petri dishesSPL10100100 mm diameter, 15 mm height
14 mm hollow punchILJIN124-0571
18 mm Ø CoverslipMarienfeld-Superior111580Circular 18 mm, thickness No. 1 (0.13 to 0.16 mm)
2% bis-acrylamide solutionBio-Rad1610142Wear protective gloves, clothing, and eye protection.
3-(Trimethoxysilyl)propyl methacrylate (TMSPMA)Sigma-Aldrich440159-500ML
3-way stopcockHyupsungHS-T-61NCAUTION: do not use if previously opened. do not resterlize or resuse
30 cm minimum volume line (for pediatric)HyupsungHS-MV-30CAUTION: do not use if previously opened. do not resterlize or resuse
35 mm cell culture dishCorning430165
40% Acrylamide SolutionBio-Rad1610140Wear protective gloves, clothing, and eye protection.
75 cm minimum volume line (for pediatric)HyupsungHS-MV-75CAUTION: do not use if previously opened. do not resterlize or resuse
acetic acidJ.T. BakerJT9508-03
Ammonium persulfate (APS)Bio-Rad1610700
Antibiotic-AntimycoticGibco15240-062
Bottom glass chipMicroFIT24 x 24 x 1 mm, custom-made, rectangular groove (6 x 12 mm, depth : 100 μm)
Collagen typeI, Rat tailCorning354236
Custom glass holderHan-Gug Mechatronicscustom-made
Dulbecco's Modified Eagle's Medium (DMEM)WelgeneLM 001-11
Dulbecco's Phosphate Buffered Saline (PBS)BiowestL0615-500w/o Magnesium, Calcium
Fetal bovine serum (FBS)Gibco26140-179
FluoSpheres amine-modified microspheresInvitrogenF87640.2 µm, yellow-green fluorescent(505/515)
Hepatocyte Growth Factor (HGF)Sigma-AldrichH1404-5UGrecombinant, human
JuLI stage live cell imaging systemNanoEnTek InAutomated X-Y-Z stage and fluorsent imaging Incubator-compatible (37 °C and 5% CO2)
Madin-Darby Canine Kidney (MDCK) celltype II
Oxygen plasma systemFemto ScienceCUTE-MPR
Pluronic F-127Sigma-AldrichP2443-250G
Rhodamine B isothiocyanate–dextranSigma-AldrichR9379-100MG70 kDa, used to estimate spatiotemporal distribution of HGF in the microfluidic channel
Steril hypodermic needle 18 GKOVAXTrim the tip of the needle and bend it 90 degrees for connecting in/out ports with volume line
Sticky tape3M/Scotch810D33 m x 19 mm
SU-8 master moldsMicroFIT4” diameter, custom-made
sulfosuccinimidyl 6-(4’-azido-2’-nitrophenylamino)hexanoate (Sulfo-SANPAH)Thermo Scientific22589Store at -20°C. Store protected from moisture and light.
Sylgard 184 Elastomer KitDow CorningPDMS
Syringe pumpChemyx Inc.model fusion 720withdraw fluid
SyringesKOVAX1, 3, 5, 10, or 50 cc for using inlet reservoir or outlet syringe pump
tetramethylethylenediamine (TEMED)Bio-Rad1610800Wear protective gloves, clothing, and eye protection.
Ultraviolet (UV) lampUVP LLC95-0248-02365 nm wavelength

References

  1. Reig, G., Pulgar, E., Concha, M. L. Cell migration: from tissue culture to embryos. Development. 141 (10), 1999-2013 (2014).
  2. Luster, A. D., Alon, R., von Andrian, U. H. Immune cell migration in inflammation: present and future t....

Explore More Articles

Traction MicroscopyMicrofluidicsCollective Cell MigrationBiochemical GradientCellular ForcesPDMSSU 8 MoldGlass Slide SilanizationPolyacrylamide Gel

This article has been published

Video Coming Soon

JoVE Logo

Privacy

Terms of Use

Policies

Research

Education

ABOUT JoVE

Copyright © 2024 MyJoVE Corporation. All rights reserved