Traction Force Microscopy in 3‐Dimensional Extracellular Matrix Networks

M. Cóndor1, J. Steinwachs2, C. Mark2, J.M. García‐Aznar1, B. Fabry2

1 Department of Mechanical Engineering, University of Zaragoza, Zaragoza, 2 Department of Physics, University of Erlangen‐Nuremberg, Erlangen
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 10.22
DOI:  10.1002/cpcb.24
Online Posting Date:  June, 2017
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Abstract

Cell migration through a three‐dimensional (3‐D) matrix depends strongly on the ability of cells to generate traction forces. To overcome the steric hindrance of the matrix, cells need to generate sufficiently high traction forces but also need to distribute these forces spatially in a migration‐promoting way. This unit describes a protocol to measure spatial maps of cell traction forces in 3‐D biopolymer networks such as collagen, fibrin, or Matrigel. Traction forces are computed from the relationship between measured force‐induced matrix deformations surrounding the cell and the known mechanical properties of the matrix. The method does not rely on knowledge of the cell surface coordinates and takes nonlinear mechanical properties of the matrix into account. © 2017 by John Wiley & Sons, Inc.

Keywords: biopolymer networks; finite elements; traction force microscopy; unconstrained force reconstruction; 3‐D cell traction forces

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Preparation of Type I Collagen Gels with Cells
  • Basic Protocol 2: Confocal Reflection Microscopy of 3‐D Collagen Gels
  • Basic Protocol 3: Unconstrained 3‐D Force Reconstruction
  • Basic Protocol 4: Macrorheology of Collagen Type I Hydrogels
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Preparation of Type I Collagen Gels with Cells

  Materials
  • T‐75 (75‐cm2) flask with cells of interest at 60% to 75% confluence
  • Complete cell culture medium, 37°C (depending the cell line): e.g., low‐glucose DMEM (1g/liter D‐glucose) with 10% FBS and 1% penicillin/streptomycin
  • 2 mg/ml, rat tail collagen (Collagen R; Matrix Bioscience)
  • 4 mg/ml bovine skin collagen (Collagen G; Matrix Bioscience)
  • 0.27 M NaHCO 3: Dissolve 2.3 g NaHCO 3 (Merck KGaA) in 100 ml distilled H 2O (store up to 1 year at 4ºC)
  • 10× DMEM medium (Biochrom)
  • 1 M NaOH: Dissolve 4 g NaOH (Sigma) in 100 ml distilled H 2O (store up to 1 year at 4ºC)
  • Dulbecco's phosphate‐buffered saline without Ca or Mg (Thermo Fisher Scientific, cat. no. 14190169)
  • 50‐ml conical polypropylene centrifuge tubes
  • 35‐mm Petri dishes
  • Aluminum or copper plate to support Petri dishes in ice bucket (∼15 × 10 cm, see Fig.  )
  • 37°C incubator, 95% relative humidity and 5% CO 2
  • Cell culture tabletop centrifuge
  • Tissue culture phase‐contrast microscope
  • Additional reagents and equipment for cell culture (unit 1.1; Phelan & May, )

Basic Protocol 2: Confocal Reflection Microscopy of 3‐D Collagen Gels

  Materials
  • Ethanol
  • Cells within collagen gel in 35‐mm Petri dishes ( protocol 1)
  • 1 mM cytochalasin D: dissolve 50 mg cytochalasin D (Sigma‐Aldrich) in 100 ml dimethyl sulfoxide (DMSO; Sigma); store up to 2 years at –20ºC
  • Complete cell culture medium, 37°C (depending the cell line): e.g., low‐glucose DMEM (1g/liter D‐glucose) with 10% FBS and 1% penicillin/streptomycin
  • Upright confocal microscope (Leica TCS SP1)
  • Lens paper
  • Argon laser 488 nm or other laser
  • Microscope environmental chamber or microscope stage incubator
  • 20× dip‐in water‐immersion objective with a high numerical aperture (NA)
  • Injection needles (optional)

Basic Protocol 3: Unconstrained 3‐D Force Reconstruction

  Materials
  • A compiled version of the SAENO software (https://github.com/Tschaul/SAENO)
  • Matlab software V.R2012b or newer (older versions of Matlab may not work with SAENO)
  • PC with Windows operating system

Basic Protocol 4: Macrorheology of Collagen Type I Hydrogels

  Materials
  • A compiled version of the analysis software CurledBeamsFit
  • The Matlab program PlotFitandData
  • Matlab software V.R2012b or newer
  • An open‐source GCC compiler for Windows, for example MinGW32
  • Computer with Windows operating system
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Figures

Videos

Literature Cited

 
  Arevalo, R. C., Urbach, J. S., & Blair, D. L. (2010). Size‐dependent rheology of type‐i collagen networks. Biophysical Journal, 99, L65–L67. doi: 10.1016/j.bpj.2010.08.008.
  Butler, J. P., Tolic‐Norrelykke, I. M., Fabry, B., & Fredberg, J. J. (2002). Traction fields, moments, and strain energy that cells exert on their surroundings. American Journal of Physiology‐Cell Physiology, 282, C595–C605. doi: 10.1152/ajpcell.00270.2001.
  Dembo, M., & Wang, Y. L. (1999). Stresses at the cell‐to‐substrate interface during locomotion of fibroblasts. Biophysical Journal, 76, 2307–2316. doi: 10.1016/S0006‐3495(99)77386‐8.
  Franck, C., Maskarinec, S. A., Tirrell, D. A., & Ravichandran, G. (2011). Three‐dimensional traction force microscopy: A new tool for quantifying cell‐matrix interactions. PLoS One, 6, e17833. doi: 10.1371/journal.pone.0017833.
  Legant, W. R., Miller, J. S., Blakely, B. L., Cohen, D. M., Genin, G. M., & Chen, C. S. (2010). Measurement of mechanical tractions exerted by cells in three‐dimensional matrices. Nature Methods, 7, 969–U113. doi: 10.1038/nmeth.1531.
  Licup, A. J., Munster, S., Sharma, A., Sheinman, M., Jawerth, L. M., Fabry, B., … MacKintosh, F. C. (2015). Stress controls the mechanics of collagen networks. Proceedings of the National Academy of Sciences of the United States of America, 112, 9573–9578. doi: 10.1073/pnas.1504258112.
  Miron‐Mendoza, M., Seemann, J., & Grinnell, F. (2010). The differential regulation of cell motile activity through matrix stiffness and porosity in three dimensional collagen matrices. Biomaterials, 31, 6425–6435. doi: 10.1016/j.biomaterials.2010.04.064.
  Palacio, J., Jorge‐Penas, A., Munoz‐Barrutia, A., Ortiz‐de‐Solorzano, C., de Juan‐Pardo, E., & Garcia‐Aznar, J. M. (2013). Numerical estimation of 3D mechanical forces exerted by cells on non‐linear materials. Journal of Biomechanics, 46, 50–55. doi: 10.1016/j.jbiomech.2012.10.009.
  Phelan, K., & May, K.M. (2015). Basic techniques in mammalian cell tissue culture. Current Protocols in Cell Biology, 66, 1.1.1‐1.1.22. doi: 10.1002/0471143030.cb0101s66.
  Sabass, B., Gardel, M. L., Waterman, C. M., & Schwarz, U. S. (2008). High resolution traction force microscopy based on experimental and computational advances. Biophysical Journal, 94, 207–220. doi: 10.1529/biophysj.107.113670.
  Steinwachs, J., Metzner, C., Skodzek, K., Lang, N., Thievessen, I., Mark, C., … Fabry, B. (2016). Three‐dimensional force microscopy of cells in biopolymer networks. Nature Methods, 13, 171–176. doi: 10.1038/nmeth.3685.
  Vader, D., Kabla, A., Weitz, D., & Mahadevan, L. (2009). Strain‐induced alignment in collagen gels. Plos One, 4, e5902. doi: 10.1371/journal.pone.0005902.
  Yang, Y. L., & Kaufman, L. J. (2009). Rheology and confocal reflectance microscopy as probes of mechanical properties and structure during collagen and collagen/hyaluronan self‐assembly. Biophysical Journal, 96, 1566–1585. doi: 10.1016/j.bpj.2008.10.063.
Internet Resources
  https://github.com/Tschaul/SAENO
  The source code of the algorithm SAENO can be downloaded free of charge from the collaborative coding platform GitHub at the above URL. A compiled version of the software and a tutorial are provided as Supplementary Software, together with a sample data set (http://lpmt.biomed.uni‐erlangen.de/3DTractions/SampleData.rar).
  https://github.com/Tschaul/Macrorheology
  The macrorheology software and a sample data set of two collagen rheology experiments can be downloaded from the GitHub platform at the above URL.
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