Generation of 3D Collagen Gels with Controlled Diverse Architectures

Andrew D. Doyle1

1 National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda Maryland
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 10.20
DOI:  10.1002/cpcb.9
Online Posting Date:  September, 2016
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Abstract

Rat tail collagen solutions have been used as polymerizable in vitro three dimensional (3D) extracellular matrix (ECM) gels for single and collective cell migration assays as well as spheroid formation. Factors such as ECM concentration, pH, ionic concentration, and temperature can alter collagen polymerization and ECM architecture. This unit describes how to generate 3D collagen gels that have distinct architectures ranging from a highly reticular meshwork of short thin fibrils with small pores to a loose matrix consisting of stiff, parallel‐bundled long fibrils by changing collagen polymerization temperature. This permits analysis of 3D cell migration in different ECM architectures found in vivo while maintaining a similar ECM concentration. Also included are collagen labeling techniques helpful for ECM visualization during live fluorescence imaging. © 2016 by John Wiley & Sons, Inc.

Keywords: ECM architecture; fluorescent labelling; rat‐tail collagen; 3D matrix

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Polymerization of Rat Tail Collagen at Different Temperatures
  • Support Protocol 1: Direct Labeling of Rat Tail Collagen Gels with NHS Ester Dyes to Visualize ECM Architecture
  • Support Protocol 2: Addition of Cells to Neutralized Collagen Solutions for 3D Culture
  • Basic Protocol 2: Labeling Rat Tail Collagen Gels with NHS Ester Dyes for Use with Live Cells
  • Support Protocol 3: Concentration of Collagen Solutions Using Salt Precipitation
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Polymerization of Rat Tail Collagen at Different Temperatures

  Materials
  • 6 mg/ml stock solution of rat tail collagen in 20 mM acetic acid
  • 10× DMEM (see recipe)
  • 10× reconstitution buffer (see recipe)
  • 1 N NaOH (see recipe)
  • 1 N HCl
  • Dulbecco's PBS with calcium and magnesium (DPBS++), 4°C and room temperature
  • Rectangular ice bucket with ice
  • 1.5‐ml microcentrifuge tubes
  • 4 × 8–in. rectangular glazed ceramic tile (optional; e.g., from local hardware store)
  • 37°C cell culture incubator
  • Echotherm chilling/heating dry bath (Torrey Pines Scientific) or standard dry heating block in a cold room (4°C)
  • 35‐mm glass‐bottom dishes (no. 1.5 coverslip, 20‐mm opening; MatTek, cat. no. P35G‐1.5‐20‐C)
  • 10‐, 100‐, and 1000‐µl positive‐displacement pipets and tips (Gilson)
  • ColorpHast pH indicator strips (range 6.5‐10, EMD Biosciences)
  • Parafilm

Support Protocol 1: Direct Labeling of Rat Tail Collagen Gels with NHS Ester Dyes to Visualize ECM Architecture

  Materials
  • 35‐mm dishes with collagen gel (see protocol 1)
  • 1 mg/ml Atto‐488 NHS ester dye (see recipe)
  • 50 mM borate buffer, pH 9.0 (see recipe)
  • Dulbecco's PBS with calcium and magnesium (DPBS++)
  • 15‐ml conical tubes

Support Protocol 2: Addition of Cells to Neutralized Collagen Solutions for 3D Culture

  Additional Materials (also see protocol 1)
  • ≤1.0 × 106 cells (e.g., human dermal fibroblasts, HT‐1080 cells) in a tissue culture dish
  • Appropriate cell culture medium
  • Centrifuge with swinging bucket rotor
  • Additional reagents and equipment for detaching cells (e.g., trypsin/EDTA solution) and counting cells

Basic Protocol 2: Labeling Rat Tail Collagen Gels with NHS Ester Dyes for Use with Live Cells

  Materials
  • 6 mg/ml stock solution of rat tail collagen in 20 mM acetic acid
  • 10× DMEM (see recipe)
  • 10× reconstitution buffer (see recipe)
  • 1 N NaOH (see recipe)
  • 1 N HCl
  • Dulbecco's PBS with calcium and magnesium (DPBS++), 4°C
  • 50 mM borate buffer, pH 9.0 (see recipe)
  • 5 mg/ml Atto‐488 NHS ester dye (see recipe)
  • 50 mM Tris buffer, pH 7.5 (from commercial 1 M stock, e.g., KD Medical)
  • 500 and 20 mM acetic acid, 4°C
  • 10‐cm tissue culture dish
  • Cell scrapers (Costar, cat. no. 3008)
  • Aluminum foil
  • Rocker in a cold room
  • 1000‐µl positive‐displacement pipet and tips (Gilson)
  • Scintillation vial (or 50‐ml beaker)
  • Large beaker (for 2‐4 liters solution)
  • 3‐ml Slide‐A‐Lyzer G2 dialysis cassette with 20,000 MW cutoff (Pierce)
  • Plastic wrap
  • 1.5‐ml microcentrifuge tubes
  • Sircol Collagen Assay kit (Accurate Chemical & Scientific)
  • 1.5‐ml conical tube

Support Protocol 3: Concentration of Collagen Solutions Using Salt Precipitation

  Materials
  • Collagen solution to be concentrated (see protocol 4)
  • 8% (w/v) NaCl solution (see recipe)
  • 500 mM acetic acid
  • 15‐ml conical centrifuge tube
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Figures

Videos

Literature Cited

Literature Cited
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