Preparation of Extracellular Matrices Produced by Cultured and Primary Fibroblasts

Janusz Franco‐Barraza1, Dorothy A. Beacham1, Michael D. Amatangelo1, Edna Cukierman1

1 Fox Chase Cancer Center, Philadelphia, Pennsylvania
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 10.9
DOI:  10.1002/cpcb.2
Online Posting Date:  June, 2016
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Abstract

Fibroblasts secrete and organize extracellular matrix (ECM), which provides structural support for their adhesion, migration, and tissue organization, besides regulating cellular functions such as growth and survival. Cell‐to‐matrix interactions are vital for vertebrate development. Disorders in these processes have been associated with fibrosis, developmental malformations, cancer, and other diseases. This unit describes a method for preparing a three‐dimensional matrix derived from fibroblastic cells; the matrix is three‐dimensional, cell and debris free, and attached to a two‐dimensional culture surface. Cell adhesion and spreading are normal on these matrices. This matrix can also be compressed into a two‐dimensional matrix and solubilized to study the matrix biochemically. © 2016 by John Wiley & Sons, Inc.

Keywords: cell‐derived extracellular matrices; primary fibroblasts; myofibroblasts; 3D cell culture; tumor‐ or cancer‐associated fibroblasts; desmoplasia

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Preparation of Extracellular Matrices Produced by Cultured or Primary Fibroblasts
  • Support Protocol 1: Phenotypic Evaluation of Unextracted ECM Cultures
  • Support Protocol 2: Fixing Extracted Matrices for Lack of Pliability Analyses
  • Assessing the Quality and Functionality of Extracted Fibroblast‐Derived Three‐Dimensional Matrices
  • Support Protocol 3: Cell Attachment Assay
  • Support Protocol 4: Determination of Cell Shape
  • Support Protocol 5: Evaluation of α‐SMA Phenotype
  • Support Protocol 6: Lysis of Re‐Plated Fibroblasts for Immunoblot Analyses
  • Preparation of Two‐Dimensional Controls
  • Support Protocol 7: Mechanical Compression of the Fibroblast‐Derived 3‐D Matrix
  • Support Protocol 8: Solubilization of Fibroblast‐Derived 3‐D Matrix
  • Isolation of Primary Fibroblasts from Fresh Tissue Samples
  • Support Protocol 9: Isolation of Primary Fibroblasts by Non‐Enzymatic Procedure
  • Support Protocol 10: Isolation of Primary Fibroblasts by Enzymatic Tissue Digestion
  • Reagents and Solutions
  • Commentary
  • Figures
     
 
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Materials

Basic Protocol 1: Preparation of Extracellular Matrices Produced by Cultured or Primary Fibroblasts

  Materials
  • NIH‐3T3 cells (ATCC) or primary fibroblasts, preferentially between passages 2 and 6 (see Support Protocols protocol 98 and protocol 109)
  • Confluent medium with fetal bovine serum (FBS; see recipe)
  • 0.25% (w/v) trypsin/0.03% (w/v) EDTA solution (see recipe)
  • 0.2% (w/v) gelatin solution (see recipe)
  • Ethanol (absolute)
  • Dulbecco's phosphate‐buffered saline with Ca++ and Mg++ (DPBS+; appendix 2A)
  • DPBS, Ca++ and Mg++ free (DPBS, appendix 2a)
  • 1% (v/v) glutaraldehyde in DPBS+ (see recipe)
  • 1 M ethanolamine (see recipe)
  • Matrix medium with ascorbic acid (see recipe)
  • Extraction buffer (see recipe), 37°C
  • 10 U/ml DNase I (Roche) in DPBS+, optional
  • Penicillin/streptomycin (Invitrogen)
  • Fungizone (amphotericin B; Invitrogen)
  • 37°C, 10% CO 2 humidified incubator
  • 15‐cm dishes (or 75‐cm2 culture flasks)
  • Inverted phase‐contrast microscope
  • 6‐well tissue culture plates or 35‐mm dishes (Corning), for matrix production
  • 22‐, 18‐, 12‐, 7‐, or 5‐mm circular high‐quality coverslips (Carolina; optional)
  • Bacterial 6‐, 12‐, 24‐, or 48‐ multi‐well petri plates for preparing matrices on coverslips, optional
  • Parafilm strips
  • Small, sterile fine‐pointed tweezers (e.g., Dumont no. 4), optional

Support Protocol 1: Phenotypic Evaluation of Unextracted ECM Cultures

  Materials
  • Fibroblast‐derived 3‐D matrix produced onto 12‐mm no. 1.0 coverslips (see protocol 1Basic Protocol)
  • Dulbecco's phosphate‐buffered saline with Ca++ and Mg++ (DPBS+; appendix 2A)
  • Fixing/permeabilization solution (see recipe)
  • Fixing solution (see recipe)
  • DPBS+ with Tween‐20 (DPBS‐T; see recipe)
  • Odyssey Blocking Buffer (LI‐COR Biosciences, P/N 927‐40000) with 1% donkey serum (see )
  • 100% Donkey serum stock (Jackson ImmunoResearch Laboratories)
  • Primary antibody cocktail: anti‐α‐Smooth Muscle Actin (α‐SMA) mouse antibody (1:300) (Sigma‐Aldrich, cat. no. A2547), anti‐fibronectin rabbit antibody (1:200) (for murine samples use Abcam, cat. no. ab23750; for human samples use Sigma, cat. no. F3648); antibodies are diluted in Odyssey Blocking Buffer
  • Secondary antibody cocktail: anti‐rabbit Cy5‐conjugated and anti‐mouse Rhodamine‐red‐conjugated affinity purified F (ab′)2 donkey fragments (both 1:100) (Jackson ImmunoResearch Laboratories, cat. no. 54557 and 54831, respectively); antibodies are diluted in Odyssey Blocking Buffer
  • SYBR green reagent for nuclei labelling (Invitrogen, cat. no. S7567): 1:10,000 dilution
  • Double‐distilled water
  • Prolong Gold anti‐fade reagent (Invitrogen)
  • Light‐protected humidified chamber (i.e., dark plastic box)
  • Parafilm squares (a minimum of two)
  • Small fine‐pointed tweezers (e.g., Dumont no. 4)
  • Paper towels
  • 24‐well plates
  • Glass microscope slides
  • Confocal microscope (objectives 40× or 60×), equipped with a Krypton/Argon laser that includes three lines, 488, 568, and 647 nm, for fluorescence excitation of dye‐labeled samples (e.g., Confocal spinning disk Ultraview, Perkin‐Elmer Life Sciences), complemented with CCD camera and image acquisition software (e.g., Velocity 6.3.0, Perkin‐Elmer Life Sciences) for 8‐16 bit images acquisition; alternatively, any microscope equipped with epi‐fluorescent capabilities, excitation and emission filters to match the above‐mentioned fluorophores, and a digital camera capable of acquiring monochromatic 8‐16 bit images can be used
  • Image analysis/edition software: ImageJ (http://imagej.nih.gov/ij/)
  • MetaMorph (Molecular Devices), Optional
  • Photoshop (Adobe), Optional
  • ImageJ OrientationJ plugin (http://bigwww.epfl.ch/demo/orientation/)
  • Excel software (Microsoft)

Support Protocol 2: Fixing Extracted Matrices for Lack of Pliability Analyses

  Materials
  • 1% (v/v) glutaraldehyde in DPBS+ (see recipe)
  • Dulbecco's phosphate‐buffered saline with Ca++ and Mg++ (DPBS+; appendix 2A)
  • 1 M ethanolamine (see recipe)
  • Penicillin/streptomycin
  • Fungizone
  • Tissue culture dishes or coverslips with matrix
  • Parafilm

Support Protocol 3: Cell Attachment Assay

  Materials
  • Semi‐confluent fibroblasts (human or mouse) in a 15‐cm dish
  • Confluent medium with fetal bovine serum (see recipe)
  • Hoechst 33342 stock solution (see recipe)
  • Dulbecco's phosphate‐buffered saline with Ca++ and Mg++ (DPBS+; appendix 2A), at both 4°C and room temperature
  • DPBS, Ca++ and Mg++ free (DPBS; appendix 2A)
  • Trypsin/EDTA solution (see recipe)
  • Glass‐bottom no. 1.5 dishes (MatTek Corporation): three containing fibroblast‐derived 3‐D matrix (see protocol 1Basic Protocol) and three with precoated 2‐D fibronectin (see )
  • Fixing solution (see recipe)
  • 15‐ml polypropylene conical tubes
  • Tissue culture centrifuge equipped with rotor suitable for conical 15‐ml tubes
  • Fluorescence inverted microscope equipped with an appropriate CCD camera and set of filters to visualize Hoechst 33342 (see Herman, , appendix 1E)
  • Image analysis software capable of counting objects (optional)

Support Protocol 4: Determination of Cell Shape

  Materials
  • Fibroblast‐derived 3‐D matrix‐covered coverslips (see protocol 1Basic Protocol)
  • Fibronectin 2‐D matrix‐coated coverslips (see recipe)
  • 2% (w/v) heat‐denatured BSA (see recipe)
  • Dulbecco's phosphate‐buffered saline with Ca++ and Mg++ (DPBS+; appendix 2A)
  • Semi‐confluent 15‐cm dish of fibroblasts (see protocol 1Basic Protocol, steps 1‐5)
  • Trypsin/EDTA solution (see recipe)
  • Confluent medium with fetal bovine serum (see recipe)
  • 1,1′‐dioctadecyl‐3,3,3′,3′‐tetramethylindocarbocyanine perchlorate (DiI) stock solution (see recipe)
  • Fixing solution (see recipe)
  • Prolong Gold mounting medium (Invitrogen)
  • 35‐mm tissue culture dishes or 6‐well plates
  • Inverted microscope
  • 15‐ml polypropylene conical tubes
  • Tissue culture centrifuge equipped with rotor suitable for 15‐ml conical tubes
  • Fine‐point forceps (e.g., Dumont 4)
  • Glass microscope slides
  • Fluorescent microscope equipped with digital camera
  • Image analysis software capable of measuring elliptical Fourier parameters

Support Protocol 5: Evaluation of α‐SMA Phenotype

  Materials
  • Fibroblast‐derived 3‐D matrix‐covered 12‐mm no. 1.0 coverslips (see protocol 1Basic Protocol)
  • 2% (w/v) heat‐denatured BSA (see recipe)
  • Dulbecco's phosphate‐buffered saline with Ca++ and Mg++ (DPBS+; appendix 2A)
  • DPBS, Ca++ and Mg++ free (DPBS; appendix 2A)
  • DPBS+ with Tween (0.05%) (DPBS+‐T; see recipe)
  • Double‐distilled water
  • Semi‐confluent 15‐cm dish of fibroblasts or 75‐cm2 flask (see protocol 1Basic Protocol, steps 1‐5)
  • Trypsin/EDTA solution (see recipe)
  • Complete medium with fetal bovine serum (see recipe)
  • Fixing solution (see recipe)
  • Fixing/Permeabilization solution (see recipe)
  • 100% Donkey serum stock (Jackson ImmunoResearch Laboratories)
  • Odyssey Blocking Buffer (LI‐COR Biosciences, P/N 927‐40000) with 1% donkey serum (see )
  • Primary antibody solution: anti‐α‐Smooth Muscle Actin (α‐SMA) mouse antibody (1:300) (Sigma‐Aldrich, cat. no. A2547) diluted in Odyssey Blocking Buffer
  • Secondary antibody solution: anti‐mouse Rhodamine‐red‐conjugated affinity purified F (ab′) 2 donkey fragment (1:100) (Jackson ImmunoResearch Laboratories, cat. no. 54831), diluted in Odyssey Blocking Buffer
  • Fluorescent labeled Phalloidin solution: 5 μl of Oregon Green Phalloidin (Life Technologies) diluted in 200 μl DPBS+ ‐Tween‐20 0.05% (see recipe)
  • Prolong Gold mounting medium (Invitrogen)
  • 24‐well culturing plates
  • Inverted microscope
  • Fine‐point forceps (e.g., Dumont 4)
  • Glass microscope slides
  • Fluorescent microscope (objectives 20× or 40×), equipped with digital camera and image acquisition software
  • Optional: Confocal microscope (objectives 40× or 60×), equipped with a Krypton/Argon laser with three lines, 488, 568, and 647 nm, for fluorescence excitation of dye‐labeled samples (e.g., Confocal spinning disk Ultraview, Perkin‐Elmer Life Sciences), complemented with CCD camera and image acquisition software (e.g., Velocity 6.3.0, Perkin‐Elmer Life Sciences) for 8‐16 bit images acquisition
  • Image analysis software, capable of measuring elliptical Fourier parameters, pixel intensity (integrated intensity) and pixel colocalization between two images:
  • ImageJ (http://imagej.nih.gov/ij/)
  • MetaMorph (Molecular Devices), optional
  • Excel software (Microsoft)

Support Protocol 6: Lysis of Re‐Plated Fibroblasts for Immunoblot Analyses

  Materials
  • Matrix‐coated ≥35‐mm dishes (see protocol 3)
  • Fibronectin‐coated ≥35‐mm dishes
  • Cell suspension from confluent cultures of fibroblasts
  • Confluent medium with fetal bovine serum (see recipe)
  • Lysis buffer (modified RIPA) reagent (see recipe) supplemented with protease and phosphatase inhibitors (see recipe), ice cold
  • Normal human or murine fibroblasts re‐plated in 3‐D matrix dishes (see protocol 1Basic Protocol, step 27)
  • Dulbecco's phosphate‐buffered saline with Ca++ and Mg++ (DPBS+; appendix 2A)
  • Dry ice/isopropanol bath
  • 5× sample buffer supplemented with 2‐mercaptoethanol
  • Anti‐FAKpY397 and anti‐total FAK (see reciperecipes)
  • Anti α‐SMA (see recipe)
  • Glutaraldehyde‐3‐phosphate dehydrogenase (GAPDH)
  • 37°C, 10% CO 2 humidified incubator
  • Cell scraper (Costar, Fisher Scientific)
  • 1.5‐ml microcentrifuge tubes (Eppendorf)
  • Sonicator (e.g., Branson Sonifier 150)
  • Scion image software beta version 4.03
  • Additional reagents and equipment for calculating the amount of 5× sample buffer supplemented with 2‐mercaptoethanol (Gallagher, , unit 6.1) and detecting proteins by immunoblotting (Gallagher et al., , unit 6.2)

Support Protocol 7: Mechanical Compression of the Fibroblast‐Derived 3‐D Matrix

  Materials
  • Superglue
  • Absolute ethanol, optional
  • Fibroblast‐derived 3‐D matrix on 22‐mm circular coverslips (see protocol 1Basic Protocol)
  • Dulbecco's phosphate‐buffered saline with Ca++ and Mg++ (DPBS+; see appendix 2A)
  • Flat platform large enough to rest on the ring (see Fig. )
  • Suitable spacer smaller in width than the diameter of the ring but longer in height than the depth of the ring (see Fig.  )
  • 12‐mm round coverslips (Carolina)
  • Teflon film: protective overlay composed of: 0.001‐in. FEP film, on 0.008‐in. vinyl film, with adhesive back (use to cover laboratory bench‐tops, Cole‐Parmer Instrument Company)
  • Cork borer (12‐mm diameter)
  • Biological hood equipped with UV light
  • Stand equipped with a horizontal ring for weight support
  • Lifting laboratory jack
  • Parafilm
  • Weight (∼158 g)
  • 35‐mm dishes
  • Inverted phase‐contrast microscope

Support Protocol 8: Solubilization of Fibroblast‐Derived 3‐D Matrix

  Materials
  • Fibroblast‐derived 3‐D matrices on 35‐mm dishes (see protocol 1Basic Protocol)
  • Solubilization reagent (see recipe)
  • Cell scraper (e.g., rubber policeman, Costar brand, Fisher Scientific)
  • 1.5‐ml microcentrifuge tubes
  • Rotator at 4°C
  • Microcentrifuge

Support Protocol 9: Isolation of Primary Fibroblasts by Non‐Enzymatic Procedure

  Materials
  • Fresh tissue samples (murine or human surgical)
  • Dulbecco's phosphate‐buffered saline with Ca++ and Mg++ (DPBS+; appendix 2A) supplemented with antibiotics (see recipe), 4°C
  • Complete fibroblastic medium with 10‐15% fetal bovine serum (FBS; see recipe)
  • Ciprofloxicin (Invitrogen), optional
  • Fungizone (amphotericin B; Invitrogen), optional
  • Trypsin/EDTA solution (see recipe)
  • BSA (Fraction V, Sigma‐Aldrich)
  • Sterile DMEM‐3%BSA‐pen/strep (see recipe)
  • 100‐mm tissue culture dishes
  • Dissecting scissors, tweezers, and scalpels (Fisher Scientific)
  • 12‐well or 6‐well tissue culture plates
  • Sterile laminar flow hood
  • 75‐cm2 or 25‐cm2 tissue culture flasks
  • Tissue culture incubator: 37°C, 5‐10% (v/v) humidified CO 2 incubator
  • Inverted phase‐contrast microscope

Support Protocol 10: Isolation of Primary Fibroblasts by Enzymatic Tissue Digestion

  Materials
  • Fresh tissue samples (murine or human surgical)
  • Sterile DMEM‐3%BSA‐pen/strep (see recipe)
  • Sterile 10× Collagenase‐3 (Worthington)
  • Complete fibroblast medium with 10% fetal bovine serum (FBS; see )
  • 37°C, 5% CO 2 incubator
  • 50‐ml polypropylene tubes
  • Centrifuge
  • 50‐μm nylon mesh (Sefar America); nylon mesh should be sterilized by autoclave
  • 100‐ and 40‐μm cell strainer (BD Falcon)
  • 75‐cm2 tissue culture flasks
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Figures

Videos

Literature Cited

  Amatangelo, M.D., Bassi, D.E., Klein‐Szanto, A.J., and Cukierman, E. 2005. Stroma‐derived three‐dimensional matrices are necessary and sufficient to promote desmoplastic differentiation of normal fibroblasts. Am. J. Pathol. 167:475‐488. doi: 10.1016/S0002‐9440(10)62991‐4.
  Buck, C.A. and Horwitz, A.F. 1987. Cell surface receptors for extracellular matrix molecules. Annu. Rev. Cell Biol. 3:179‐205. doi: 10.1146/annurev.cb.03.110187.001143.
  Burridge, K. and Chrzanowska‐Wodnicka, M. 1996. Focal adhesions, contractility, and signaling. Annu. Rev. Cell Dev. Biol. 12:463‐518. doi: 10.1146/annurev.cellbio.12.1.463.
  Cukierman, E. 2005. Cell migration analyses within fibroblast‐derived 3‐D matrices. Methods Mol. Biol. 294:79‐93.
  Cukierman, E., Pankov, R., Stevens, D.R., and Yamada, K.M. 2001. Taking cell‐matrix adhesions to the third dimension. Science 294:1708‐1712. doi: 10.1126/science.1064829.
  Friedl, P. and Brocker, E.B. 2000. The biology of cell locomotion within three‐dimensional extracellular matrix. Cell Mol. Life Sci. 57:41‐64. doi: 10.1007/s000180050498.
  Gallagher, S. 2007. One‐dimensional SDS gel electrophoresis of proteins. Curr. Protoc. Cell Biol. 37: 6.1.1‐6.1.38.
  Gallagher, S, Wintson, S.E., Fuller, S.A., and Hurrell, J.G. 2011. Immunoblotting and immunodetection. Curr. Protoc. Cell Biol. 52:6.2.1‐6.2.28.
  Geiger, B., Bershadsky, A., Pankov, R., and Yamada, K.M. 2001. Transmembrane crosstalk between the extracellular matrix and the cytoskeleton. Nat. Rev. Mol. Cell Biol. 2:793‐805. doi: 10.1038/35099066.
  Gupta, V., Bassi, D.E., Simons, J.D., Devarajan, K., Al‐Saleem, T., Uzzo, R.G., and Cukierman, E. 2011 Elevated expression of stromal palladin predicts poor clinical outcome in renal cell carcinoma. PLoS One 6:e21494. doi: 10.1371/journal.pone.0021494.
  Hay, E.D. 1991. Cell Biology of Extracellular Matrix, 2nd ed. Plenum Press, New York.
  Herman, B. 2001. Absorption and emission maxima for common fluorophores. Curr. Protoc. Cell Biol. 00:A.1E.1‐A.1E.5.
  Hynes, R.O. 1999. Cell adhesion: Old and new questions. Trends Cell Biol. 9:M33‐M77. doi: 10.1016/S0962‐8924(99)01667‐0.
  Katz, B.Z., Zamir, E., Bershadsky, A., Kam, Z., Yamada, K.M., and Geiger, B. 2000. Physical state of the extracellular matrix regulates the structure and molecular composition of cell‐matrix adhesions. Mol. Biol. Cell 11:1047‐1060. doi: 10.1091/mbc.11.3.1047.
  Melcher, K. and Chen, H.‐T. 2007. Identification and analysis of multiprotein complexes through chemical cross‐linking. Curr. Protoc. Cell Biol. 33:17.10.1‐17.10.29.
  Pankov, R., Cukierman, E., Katz, B.Z., Matsumoto, K., Lin, D.C., Lin, S., Hahn, C., and Yamada, K.M. 2000. Integrin dynamics and matrix assembly: Tensin‐dependent translocation of alpha(5)beta(1) integrins promotes early fibronectin fibrillogenesis. J. Cell Biol. 148:1075‐1090. doi: 10.1083/jcb.148.5.1075.
  Phelan, K. and May, K.M. 2015. Basic techniques in mammalian cell tissue culture. Curr. Protoc. Cell Biol. 66:1.1.1‐1.1.22.
  Rybinski, B., Franco‐Barraza, J., and Cukierman, E. 2014. The wound healing, chronic fibrosis, and cancer progression triad. Physiol. Genomics 46:223‐244. doi: 10.1152/physiolgenomics.00158.2013.
  Vlodavsky, I. 2001. Preparation of extracellular matrices produced by cultured corneal endothelial and PF‐HR9 endodermal cells. Curr. Protoc. Cell Biol. 1:10.4.1‐10.4.14.
Key References
  Cukierman et al. (2001). See above.
  The source for procedures and materials described in this unit.
  Vlodavsky, I., Lui, G.M., and Gospodarowicz, D. 1980. Morphological appearance, growth behavior and migratory activity of human tumor cells maintained on extracellular matrix versus plastic. Cell 19(3):607‐616.
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