3‐D Extracellular Matrix from Sectioned Human Tissues

Catherine B. Campbell1, Edna Cukierman2, Vira V. Artym3

1 Laboratory of Cell and Developmental Biology, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, Maryland, 2 Cancer Biology Program, Fox Chase Cancer Center, Philadelphia, Pennsylvania, 3 Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University Medical School, Washington, D.C.
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 19.16
DOI:  10.1002/0471143030.cb1916s62
Online Posting Date:  March, 2014
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Abstract

Cell adhesion, migration, and signaling in physiologically normal and pathological processes depend highly on the extracellular matrix that the cell interacts with. A variety of in vitro models of two‐dimensional and three‐dimensional extracellular matrices have been developed to study multiple aspects of cellular behavior. However, there is a profound need for in vitro models of extracellular matrices to closely mimic both biochemical and physical aspects of a three‐dimensional in vivo cellular environment. This unit outlines the preparation of human‐tissue‐derived, cell‐free, three‐dimensional extracellular matrices for studying cellular behavior and cell‐extracellular matrix interactions ex vivo. These protocols can be used to prepare cell‐free matrices from a variety of normal and cancerous tissues. This unit also provides protocols for quality control of acellular matrix preparations, and for immunostaining of cells for specific cellular proteins as well as of extracellular matrices for their components. Curr. Protoc. Cell Biol. 62:19.16.1‐19.16.20. © 2014 by John Wiley & Sons, Inc.

Keywords: three‐dimensional (3‐D) extracellular matrix; cell extraction; human tissue; acellular matrix; H&E staining; trichrome staining; immunostaining; collagen

     
 
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Table of Contents

  • Basic Protocol 1: Extraction of Human Tissue Sections to Generate a Cell‐Free 3‐D Matrix
  • Support Protocol 1: Plating of Cells, Fixation, Permeabilization, and Immunofluorescence Staining
  • Support Protocol 2: Embedding Tissue in O.C.T.
  • Support Protocol 3: Cryostat Tissue Sectioning
  • Support Protocol 4: Hematoxylin and Eosin Stain
  • Support Protocol 5: Masson's Trichrome Stain
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Extraction of Human Tissue Sections to Generate a Cell‐Free 3‐D Matrix

  Materials
  • Extraction buffer (see recipe)
  • 12‐µm thick cryostat sections of human tissue samples on positively charged slides in a slide box (see Support Protocols protocol 32 and protocol 43)
  • Phosphate‐buffered saline (PBS; appendix 2A), sterile
  • 10 U/ml DNase I recombinant, RNase‐free (Roche) in PBS (see recipe)
  • 37°C water bath
  • 100 × 15–mm plastic petri dishes (BD Falcon)
  • Orbital rocker (e.g., VWR DW‐150 Waver)
  • Glass Pasteur pipets
  • Vacuum aspirator
  • Parafilm
  • Inverted phase‐contrast microscope

Support Protocol 1: Plating of Cells, Fixation, Permeabilization, and Immunofluorescence Staining

  Materials
  • Cell culture medium containing fetal bovine serum (FBS; see recipe)
  • DMEM with glutamine and high glucose (HyClone)
  • 0.05% trypsin‐EDTA (1×) (Gibco)
  • Penicillin/streptomycin (10,000 U/ml penicillin and 10,000 µg/ml streptomycin, Gibco)
  • Extracted human tissue matrices on positively charged slides (see protocol 1Basic Protocol)
  • Cell culture of MDA‐MB‐231 cells cultured in 75‐cm2 cell‐culture flask
  • Fixation solution containing 4% paraformaldehyde (see recipe)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • 1× phosphate buffered saline with Ca2+ and Mg2+ (HyClone)
  • Permeabilization solution (see recipe)
  • Blocking solution (see recipe)
  • 0.05% Tween 20 in PBS (see recipe)
  • Primary antibody solution (see recipe)
  • Secondary antibody solution (see recipe)
  • Fluoro‐gel with anti‐fading agent (Electron Microscopy Sciences, cat. no. 17983‐20)
  • Clear nail polish
  • 37°C water bath
  • 100 × 15–mm plastic petri dishes (BD Falcon)
  • 37°C, 10% CO 2 humidified incubator
  • Aspirator
  • Inverted phase contrast microscope
  • 50‐ml plastic conical centrifuge tubes
  • Centrifuge
  • Cell counter
  • Parafilm
  • Immunostain moisture chamber (Ted Pella, cat. no. 21049)
  • Circular rocker (Waver, VWR, cat. no. DW‐150)
  • 24 × 60–mm, no. 1 Premium cover glass slips (Fisher Scientific, cat. no. 12‐548‐5P)

Support Protocol 2: Embedding Tissue in O.C.T.

  Materials
  • Powdered dry ice
  • 70% ethanol
  • Human tissue samples (e.g., pancreas or breast, available from, e.g., National Disease Research Interchange, NDRI; Cooperative Human Tissue Network, CHTN; Asterand)
  • O.C.T. compound medium (Tissue‐Tek)
  • Solid copper cylinder (2‐in.diameter, 4‐in. long)
  • Aluminum foil
  • 22 × 22–mm Peel‐A‐Way disposable plastic tissue embedding molds (Electron Microscopy Sciences, cat. no. 70182)
  • Tweezers

Support Protocol 3: Cryostat Tissue Sectioning

  Materials
  • O.C.T.‐embedded human tissue sample molds (see protocol 3)
  • O.C.T compound medium (Tissue‐Tek)
  • Cryostat (Leica CM3050 S)
  • Specimen disc (30‐mm)
  • Paint brush, soft round tip, no. 4 width
  • Tweezers (e.g., Dumont no. 5)
  • Low‐profile disposable blades (80 × 8 × 0.25–mm)
  • Plastic slide box (holds 100 slides, e.g., Ted Pella)
  • Daigger Superfrost Plus/Colorfrost Plus 75 × 25–mm pre‐cleaned microslides (positively charged)

Support Protocol 4: Hematoxylin and Eosin Stain

  Materials
  • Tissue slides at –80°C (see protocol 5)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • Hematoxylin solution, Gill no. 3 (Sigma)
  • Acid ethanol (see recipe)
  • Acidified eosin Y (see recipe)
  • 90% and 100% ethanol
  • Xylene
  • Permount histological mounting medium (Fisher Scientific)
  • Wheaton glass slide staining dishes with removable racks (Fisher Scientific)
  • 24 × 60–mm no. 1 premium cover glass slips (Fisher Scientific, cat. no. 12‐548‐5P)

Support Protocol 5: Masson's Trichrome Stain

  Materials
  • Masson's Trichrome stain kit (Thermo Scientific/Richard‐Allan Scientific product no. 010, cat. no. 87019) containing:
    • Weigert's iron hematoxylin, part A
    • Weigert's iron hematoxylin, part B
    • Biebrich scarlet‐acid fuchsin solution
    • Aniline blue stain solution
    • Phosphotungstic‐phosphomolybdic acid solution
    • Bouin's solution
  • Working Weigert's iron solution (see recipe)
  • 1% acetic acid solution
  • 100% ethanol
  • Xylene
  • Permount histological mounting medium (Fisher Scientific)
  • 56°C water bath
  • Glass staining dishes with cover (Wheaton Science Products)
  • Glass staining rack (Wheaton Science Products)
  • 24 × 60–mm no. 1 premium glass cover slips (Fisher Scientific, cat. no. 12‐548‐5P)
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Figures

Videos

Literature Cited

  Carletti, E., Motta, A., and Migliaresi, C. 2011. Scaffolds for tissue engineering and 3D cell culture. Methods Mol. Biol. 695:17‐39.
  Cukierman, E., Pankov, R., Stevens, D.R., and Yamada, K.M. 2001. Taking cell‐matrix adhesions to the third dimension. Science 294:1708‐1712.
  Doyle, A.D., Petrie, R.J., Kutys, M.L., and Yamada, K.M. 2013. Dimensions in cell migration. Curr. Opin. Cell Biol. 25:642‐649.
  Even‐Ram, S. and Yamada, K.M. 2005. Cell migration in 3D matrix. Curr. Opin. Cell Biol. 17:524‐532.
  Friedl, P. and Bröcker E.B. 2000. The biology of cell locomotion within three‐dimensional extracellular matrix. Cell. Mol. Life Sci. 57:41‐64.
  Friedl, P. and Wolf, K. 2010. Plasticity of cell migration: A multiscale tuning model. J. Cell Biol. 188:11‐19.
  Grinnell, F. and Petroll, W.M. 2010. Cell motility and mechanics in three‐dimensional collagen matrices. Annu. Rev. Cell Dev. Biol. 26:335‐361.
  Haycock, J.W. 2011. 3D cell culture: A review of current approaches and techniques. Methods Mol. Biol. 695:1‐15.
  Kutys, M., Doyle, A.D., and Yamada, K.M. 2013. Regulation of cell adhesion and migration by cell‐derived matrices. Exp. Cell Res. 319:2434‐2439.
  Ning, L., Zhang, Y., Chen, X., Luo, J., Li, X., Yang, Z., and Qin, T. 2012. Preparation and characterization of decellularized tendon slices for tendon tissue engineering. J. Biomed. Mat. Res. A 100:1448‐1456.
  Pampaloni, F., Reynaud, E.G., and Stelzer, E.H. 2007. The third dimension bridges the gap between cell culture and live tissues. Nat. Rev. Mol. Cell Biol. 8:839‐845.
  Provenzano, P.P., Inman, D.R., Eliceiri, K.W., Knittel, J.G., Yan, L., Rueden, C.T., White, J.G., and Keely, P.J. 2008. Collagen density promotes mammary tumor initiation and progression. BMC Medicine 6:11.
  Scicchitano, M.S., Dalmas, D.A., Bertiaux, M.A., Anderson, S.M., Turner, L.R., Thomas, R.A., Mirable, R., and Boyce, R.W. 2006. Preliminary comparison of quantity, quality, and microarray performance of RNA extracted from formalin‐fixed, paraffin‐embedded, and unfixed frozen tissue samples. J. Histochem. Cytochem. 54:1229‐1237.
  Takezawa, T., Takenouchi, T., Imai, K., Takahashi, T., and Hashizume, K. 2002. Cell culture on thin tissue sections commonly prepared for histopathology. FASEB J. 16:1847‐1849.
  Tibbitt, M.W. and Anseth, K.S. 2012. Dynamic microenvironments: The fourth dimension. Sci. Transl. Med. 4:160ps24.
  Vidi, P.A., Bissell, M.J., and Lelièvre, S.A. 2013. Three‐dimensional culture of human breast epithelial cells: the how and the why. Methods Mol. Biol. 945:193‐219.
  Yamada, K.M. and Cukierman, E. 2007. Modeling tissue morphogenesis and cancer in 3D. Cell 130:601‐610.
Internet Resources
  http://www.atcc.org/products/all/HTB‐26.aspx
  Website for information on MDA‐MB‐231 human breast cancer epithelial cell line.
  http://www.bcm.edu/mcb/rosenlab/index.cfm?pmid=12983
  Website for H&E protocol used as a basis and further modified.
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