Engineering Three‐Dimensional Collagen Matrices to Provide Contact Guidance during 3D Cell Migration

Paolo P. Provenzano1, Kevin W. Eliceiri1, David R. Inman1, Patricia J. Keely1

1 University of Wisconsin, Madison, Wisconsin
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 10.17
DOI:  10.1002/0471143030.cb1017s47
Online Posting Date:  June, 2010
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Abstract

Cell invasion requires that cells navigate complex three‐dimensional matrices in vivo. Topological cues are provided and three‐dimensional cell migration and invasion facilitated by the alignment of collagen fibers proximal to tumors. In order to better understand the molecular mechanisms by which cells recognize and migrate along aligned matrices, in vitro assays are needed that recapitulate topological features of the in vivo matrix. Here, we describe two approaches for creating aligned three‐dimensional collagen matrices, both dependent and independent of cell‐mediated alignment. Approaches to quantify alignment and visualize the collagen matrix relative to the cells by second‐harmonic generation are included. These assays are readily adaptable to a variety of cells and biological questions related to three‐dimensional cell migration. Curr. Protoc. Cell Biol. 47:10.17.1‐10.17.11. © 2010 by John Wiley & Sons, Inc.

Keywords: collagen alignment; three‐dimensional cell migration; invasion

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Preparation of a 3D Migration Assay Using Nested Cell‐Seeded Collagen Gels
  • Basic Protocol 2: Preparation of Aligned Collagen Matrices to Construct a 3D Migration Assay with Defined Contact Guidance Cues
  • Support Protocol 1: Imaging Collagen Matrix Alignment and Cell Migration with Multiphoton Excitation and Second‐Harmonic Generation Microscopy
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Preparation of a 3D Migration Assay Using Nested Cell‐Seeded Collagen Gels

  Materials
  • Sub‐confluent epithelial or fibroblast cells on a standard tissue culture dish/flask
  • Culture medium of choice for the chosen cell type
  • Phosphate‐buffered saline (PBS) or serum‐free culture medium
  • Trypsin/EDTA solution (Cellgro)
  • Rat tail collagen (BD Biosciences)
  • 0.1 M HEPES buffer in 2× PBS
  • 15‐ml screw‐cap conical tubes
  • 96‐well ultra‐low adhesion culture plate (Corning)
  • 37°C cell culture incubator
  • 28‐G syringe needle
  • 1000‐µl pipets with the tips cut off
  • 6‐well culture dishes (Corning ultra‐low attachment plates work very well, but other more standard plates may be substituted as needed)
  • Additional reagents and equipment for counting the cells (unit 1.1) and optical imaging ( protocol 3)

Basic Protocol 2: Preparation of Aligned Collagen Matrices to Construct a 3D Migration Assay with Defined Contact Guidance Cues

  Materials
  • Sub‐confluent epithelial or fibroblast cells on a standard tissue culture dish/flask
  • Culture medium of choice for the chosen cell type
  • Trypsin/EDTA solution (Cellgro)
  • Rat tail collagen (BD Biosciences)
  • 0.1 M HEPES buffer in 2× PBS
  • Rectangular mold (∼1‐ × 3‐in.) or Nunc multidish, 4‐well rectangular
  • 1.5‐µm diameter streptavidin‐coated iron oxide beads (Bangs Labs, cat. no. BM551)
  • Cylindrical magnet (∼2G; McMaster, cat. no. 5862K32)
  • 37°C culture incubator
  • Small spatula or cell scraper
  • Additional reagents and equipment for detaching and counting cells and preparing collage solution ( protocol 1) and optical imaging ( protocol 3)
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Figures

Videos

Literature Cited

   Condeelis, J. and Segall, J.E. 2003. Intravital imaging of cell movement in tumours. Nat. Rev. Cancer. 3:921‐930.
   Dallon, J.C., Sherratt, J.E., and Maini, P.K. 1999. Mathematical modelling of extracellular matrix dynamics using discrete cells: Fiber orientation and tissue regeneration. J. Theor. Biol. 199:449‐471.
   Denk, W., Strickler, J.H., and Webb, W.W. 1990. Two‐photon laser scanning fluorescence microscopy. Science 248:73‐76.
   Diaspro, A. and Sheppard, C.J.R. 2002. Two‐photon excitation fluorescence microscopy. In Confocal and Two‐Photon Microscopy: Foundations, Applications, and Advances. (A. Diaspro, ed.) pp. 39‐73. Wiley‐Liss, Inc., New York.
   Dickinson, R.B., Guido, S., and Tranquillo, R.T. 1994. Biased cell migration of fibroblasts exhibiting contact guidance in oriented collagen gels. Ann. Biomed. Eng. 22:342‐356.
   Doyle, A.D., Wang, F.W., Matsumoto, K., and Yamada, K.M. 2009. One‐dimensional topography underlies three‐dimensional fibrillar cell migration. J. Cell Biol. 184:481‐490.
   Friedl, P. and Gilmour, D. 2009. Collective cell migration in morphogenesis, regeneration and cancer. Nat. Rev. Mol. Cell Biol. 10:445‐457.
   Gehler, S., Baldassarre, M., Lad, Y., Leight, J.L., Wozniak, M.A., Riching, K.M., Eliceiri, K.W., Weaver, V.M., Calderwood, D.A., and Keely, P.J. 2009. Filamin A‐beta1 integrin complex tunes epithelial cell response to matrix tension. Mol. Biol. Cell 20:3224‐3238.
   Grinnell, F., Rocha, L.B., Iucu, C., Rhee, S., and Jiang, H. 2006. Nested collagen matrices: A new model to study migration of human fibroblast populations in three dimensions. Exp. Cell Res. 312:86‐94.
   Guido, S. and Tranquillo, R.T. 1993. A methodology for the systematic and quantitative study of cell contact guidance in oriented collagen gels. Correlation of fibroblast orientation and gel birefringence. J. Cell Sci. 105:317‐331.
   Guo, C. and Kaufman, L.J. 2007. Flow and magnetic field induced collagen alignment. Biomaterials. 28:1105‐1114.
   Hegerfeldt, Y., Tusch, M., Brocker, E.B., and Friedl, P. 2002. Collective cell movement in primary melanoma explants: Plasticity of cell‐cell interaction, beta1‐integrin function, and migration strategies. Cancer Res. 62:2125‐2130.
   Helmchen, F. and Denk, W. 2005. Deep tissue two‐photon microscopy. Nat. Methods 2:932‐940.
   Ingman, W.V., Wyckoff, J., Gouon‐Evans, V., Condeelis, J., and Pollard, J.W. 2006. Macrophages promote collagen fibrillogenesis around terminal end buds of the developing mammary gland. Dev. Dyn. 235:3222‐3229.
   Lauffenburger, D.A. and Horwitz, A.F. 1996. Cell migration: A physically integrated molecular process. Cell 84:359‐369.
   Lee, P.F., Yeh, A.T., and Bayless, K.J. 2009. Nonlinear optical microscopy reveals invading endothelial cells anisotropically alter three‐dimensional collagen matrices. Exp. Cell Res. 315:396‐410.
   Miron‐Mendoza, M., Seemann, J., and Grinnell, F. 2008. Collagen fibril flow and tissue translocation coupled to fibroblast migration in 3D collagen matrices. Mol. Biol. Cell 19:2051‐2058.
   Mohler, W., Millard, A.C., and Campagnola, P.J. 2003. Second harmonic generation imaging of endogenous structural proteins. Methods 29:97‐109.
   Petrie, R.J., Doyle, A.D., and Yamada, K.M. 2009. Random versus directionally persistent cell migration. Nat. Rev. Mol. Cell Biol. 10:538‐549.
   Provenzano, P.P., Eliceiri, K.W., Campbell, J.M., Inman, D.R., White, J.G., and Keely, P.J. 2006. Collagen reorganization at the tumor‐stromal interface facilitates local invasion. BMC Med. 4:38.
   Provenzano, P.P., Inman, D.R., Eliceiri, K.W., Trier, S.M., and Keely, P.J. 2008a. Contact guidance mediated three‐dimensional cell migration is regulated by Rho/ROCK‐dependent matrix reorganization. Biophys. J. 95:5374‐5384.
   Provenzano, P.P., Rueden, C.T., Trier, S.M., Yan, L., Ponik, S.M., Inman, D.R., Keely, P.J., and Eliceir, K.Wi. 2008b. Nonlinear optical imaging and spectral‐lifetime computational analysis of endogenous and exogenous fluorophores in breast cancer. J. Biomed. Opt. 13:031220.
   Provenzano, P.P., Eliceiri, K.W., and Keely, P.J. 2009a. Multiphoton microscopy and fluorescence lifetime imaging microscopy (FLIM) to monitor metastasis and the tumor microenvironment. Clin. Exp. Metastasis. 26:357‐370.
   Provenzano, P.P., Inman, D.R., Eliceiri, K.W., and Keely, P.J. 2009b. Matrix density‐induced mechanoregulation of breast cell phenotype, signaling, and gene expression through a FAK‐ERK linkage. Oncogene. 10:4326‐4343.
   Teixeira, A.I., Abrams, G.A., Bertics, P.J., Murphy, C.J., and Nealey, P.F. 2003. Epithelial contact guidance on well‐defined micro‐ and nanostructured substrates. J. Cell Sci. 116:1881‐1892.
   Zipfel, W.R., Williams, R.M., and Webb, W.W. 2003. Nonlinear magic: Multiphoton microscopy in the biosciences. Nat. Biotechnol. 21:1369‐1377.
Key References
   Provenzano, P.P. et al. 2008a. See above
  The source of procedures described in this unit.
   Guo, C. and L.J. Kaufman. 2007. See above.
  The original work from which the protocol for aligning collagen with magnetic beads was adapted.
   Miron‐Mendoza, M. et al. 2008. See above.
  This study describes the use of nested matrices to study fibroblast migration in collagen matrices.
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