Imaging Cells in Three‐Dimensional Collagen Matrix

Vira V. Artym1, Kazue Matsumoto2

1 Lombardi Comprehensive Cancer Center, Georgetown University, Washington, D.C., 2 Laboratory of Cell and Developmental Biology, NIDCR, NIH, Bethesda, Maryland
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 ( protocol 3)
  • 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 ( protocol 2)
  • 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% CO 2 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 ( 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

Videos

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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