Imaging Cells in Three‐Dimensional Collagen Matrix

Vira V. Artym1,2, Kazue Matsumoto1

1 Laboratory of Cell and Developmental Biology, NIDCR, NIH, Bethesda, Maryland, 2 Lombardi Comprehensive Cancer Center, Georgetown University, Washington, D.C.
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 10.18
DOI:  10.1002/0471143030.cb1018s48
Online Posting Date:  September, 2010
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Abstract

The use of in vitro three-dimensional (3-D) collagen matrices to mimic an in vivo cellular environment has become increasingly popular and is broadening our understanding of cellular processes and cell-ECM interactions. To study cells in in vitro 3-D collagen matrices, both cellular proteins and the collagen matrix must be visualized. In this unit, the authors describe the protocol and provide troubleshooting for immunolabeling of cells in 3-D collagen gels to localize and visualize cellular proteins with high-resolution fluorescence confocal microscopy. The authors then describe confocal reflection microscopy as a technique for direct imaging of 3-D fibrillar collagen matrices by discussing the advantages and disadvantages of the technique. They also provide instrument settings required for simultaneous imaging of cellular proteins with fluorescence confocal imaging and 3-D collagen fibrils with confocal reflection microscopy. Additionally, the authors provide protocols for a “cell sandwiching” technique to prepare cell cultures in 3-D collagen matrices required for high-resolution confocal imaging. Curr. Protoc. Cell Biol. 48:10.18.1-10.18.20. © 2010 by John Wiley & Sons, Inc.

Keywords: three-dimensional collagen matrix; collagen type I; Nutragen; confocal reflection microscopy; immunostaining of three-dimensional cell samples; invasion

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Immunolabeling Cells in Three-Dimensional Collagen Matrices for Immunofluorescence Microscopy
  • Support Protocol 1: Collagen Preparation for Three-Dimensional Collagen Matrices
  • Support Protocol 2: Polymerization of Collagen Three-Dimensional Gels and Preparation of Three-Dimensional Cell Culture
  • Basic Protocol 2: Simultaneous Fluorescence Confocal Imaging of Cells and Confocal Reflection Imaging of Collagen in In Vitro Three-Dimensional Gels
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Immunolabeling Cells in Three-Dimensional Collagen Matrices for Immunofluorescence Microscopy

 Materials
  • 4% (w/v) paraformaldehyde/5% (w/v) sucrose in PBS (see recipe)
  • Sample of cells in 3-D collagen gel polymerized in LabTek 8-well chambered coverglass (Support Protocol 2)
  • Phosphate-buffered saline (PBS; appendix 2A), filtered through a 0.22-µm filter
  • 0.5% (v/v) Triton X-100 in PBS (see recipe)
  • Blocking solution (see recipe)
  • PBS/Tween (see recipe)
  • Primary antibody solution (see recipe)
  • Secondary antibody solution (see recipe)
  • Additional reagents and equipment for confocal microscopy (unit 4.5)

Support Protocol 1: Collagen Preparation for Three-Dimensional Collagen Matrices

 Materials
  • 10× DMEM with phenol red (see recipe)
  • 10× reconstitution buffer (see recipe)
  • Phosphate-buffered saline (PBS; appendix 2A), filtered through a 0.22-µm filter
  • 2 N NaOH
  • 2 N HCl
  • 9.44 mg/ml solution of collagen type I in 0.02 N hydrochloric acid (from BD Biosciences)
  • ColorpHast pH-indicator strips with pH range 6.5–10.0 (EMD Biosciences, http://www.emdchemicals.com/)

Support Protocol 2: Polymerization of Collagen Three-Dimensional Gels and Preparation of Three-Dimensional Cell Culture

 Materials
  • Neutralized solution of rat tail collagen diluted to a final concentration of 2.5 mg/ml (Support Protocol 1)
  • Cell culture medium
  • Cell suspension (e.g., MDA-MB-231 breast cell carcinoma line; ATCC no. HTB-26) at 1.25 × 104 cells/ml in cell culture medium
  • LabTek 8-well chambered coverglass (borosilicate no. 1.0 coverglass; Nunc, cat. no. 155411)
  • 37°C, 10% CO2 cell culture incubator
  • Tissue culture microscope with 10× objective and phase-contrast optics

Basic Protocol 2: Simultaneous Fluorescence Confocal Imaging of Cells and Confocal Reflection Imaging of Collagen in In Vitro Three-Dimensional Gels

 Materials
  • Immersion oil
  • Sample of cells in three-dimensional collagen gel polymerized in LabTek 8-well chambered coverglass and immunostained for proteins of interest (Basic Protocol 1)
  • Inverted confocal microscope equipped with high NA 63× and/or 100× oil immersion objectives (also see unit 4.5)
  • Emission filters for specific emission wavelength of chromophores conjugated to anti-bodies used for immunostaining (Chroma, Omega, or available from confocal microscope manufacturer)
  • Image analysis software
  • Additional reagents and equipment for confocal microscopy (unit 4.5)
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Figures

  •  FigureFigure 10.18.1 Comparing fluorescence and reflection confocal microscopy for imaging of three-dimensional collagen sandwiches. (A) Assembly of a 3-D collagen sandwich involves polymerization of first collagen layer on glass surface, plating of the cells on this collagen layer, and (B) polymerization of the second collagen layer to encapsulate cells in a 3-D environment. The authors used collagen of two colors: one labeled with AlexaFluor 568 (in green) and another labeled with AlexaFluor 647 (in red) to demonstrate cell sandwiching between two collagen layers. Fluorescence microscopy was employed to visualize both collagen layers labeled with fluorescent AlexaFluor dyes (green and red). The collagen from the second layer penetrates into the first layer and stitches both layers together. The overlay of both layers is seen as yellow when green and red fluorescent dyes colocalize. Reflection confocal microscopy was used to visualize the collagen matrix of the sandwich (in blue). Cell nuclei were labeled with Hoechst (in magenta). (C) A series of X-Y slices through 3-D collagen sandwich taken at different Z-distances from the glass surface. Light reflected and scattered by the glass coverslip obscures images of collagen 3 µm in proximity to the glass surface in reflection confocal microscopy (Z3). Scale bar = 28 µm. (D) Light reflected from the cellular membranes contributes the cell outline to the image of collagen fibers (arrows).
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  •  FigureFigure 10.18.2 Morphology of collagen fibers in three-dimensional collagen sandwich. The authors used collagen labeled with AlexaFluor 647 (in red) to polymerize the first collagen layer, and collagen labeled with AlexaFluor 568 (in green) to polymerize the second collagen layer of a 3-D sandwich. X-Y slices taken at different Z-distances from the glass demonstrate that collagen from the second layer penetrates into the collagen of the first layer (A) and initiates collagen fibrils for the second layer of the three-dimensional collagen sandwich (B). The collagen fibers close to the glass surface appear straight (A and B), possibly from experiencing the tension from the stiffness of the glass. The collagen fibers of the second layer at a distance of about 30 µm and greater from the glass surface appear more wavy and relaxed (C and D). Unit size for the XYZ projection is 10 µm.
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  •  FigureFigure 10.18.3 Configuration for fluorescence imaging with 488-nm excitation for three-channel simultaneous fluorescence, and reflection confocal microscopy with the Zeiss510 NLO imaging system.
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  •  FigureFigure 10.18.4 Configuration for reflection imaging with 543-nm excitation for three-channel simultaneous fluorescence, and reflection confocal microscopy with the Zeiss510 NLO imaging system.
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  •  FigureFigure 10.18.5 Configuration for fluorescence imaging with 633-nm excitation for three-channel simultaneous fluorescence, and reflection confocal microscopy with the Zeiss510 NLO imaging system.
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  •  FigureFigure 10.18.6 Breast carcinoma cells transmigrating through the three-dimensional collagen matrix. The actin cytoskeleton was labeled with phalloidin-AlexaFluor 488 (green) and the sites of tyrosine phosphorylation were labeled with anti-phospho-tyrosine primary antibody, followed by secondary Cy5-conjugated antibody (red). Collagen matrix was imaged with reflection confocal microscopy (blue). Images were acquired with a 63× 1.4 NA oil objective. Volocity software was used for image presentation as XYZ projections (A) and extended focus (B). Scale bars = 19 µm.
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Literature Cited

Literature Cited
    Even-Ram, S. and Yamada, K.M. 2005. Cell migration in three-dimensional matrix. Curr. Opin. Cell Biol. 17:524-532.
    Eyre, D.R., Paz, M.A., and Gallop, P.M. 1984. Cross-linking in collagen and elastin. Annu. Rev. Biochem. 53:717-748.
    Gelman, R.A., Williams, B.R., and Piez, K.A. 1979. Collagen fibril formation: Evidence for a multistep process. J. Biol. Chem. 254:180-186.
    Packard, B.Z., Artym, V.V. Komoriya, A., and Yamada, K.M. 2009. Direct visualization of protease activity on cells migrating in three-dimensions. Matrix Biol. 28:3-10.
    Pedersen, J.A. and Swartz, M.A. 2005. Mechanobiology in the third dimension. Ann. Biomed. Eng. 33:1469-1490.
    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.
    Sabeh, F., Shimizu-Hirota, R., and Weiss, S.J. 2009. Protease-dependent versus -independent cancer cell invasion programs: Three-dimensional amoeboid movement revisited. J. Cell Biol. 185:11-19.
    Schindler, M., Nur, E.K.A., Ahmed, I., Kamal, J., Liu, H.Y., Amor, N., Ponery, A.S., Crockett, D.P., Grafe, T.H., Chung, H.Y., Weik, T., Jones, E., and Meiners, S. 2006. Living in three dimensions: Three-dimensional nanostructured environments for cell culture and regenerative medicine. Cell Biochem. Biophys. 45:215-227.
    Williams, B.R., Gelman, R.A., Poppke, D.C., and Piez, K.A. 1978. Collagen fibril formation: Optimal in vitro conditions and preliminary kinetic results. J. Biol. Chem. 253:6578-6585.
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