Preparation of Hydrogel Substrates with Tunable Mechanical Properties

Justin R. Tse1, Adam J. Engler1

1 Department of Bioengineering, University of California, San Diego, La Jolla, California
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 10.16
DOI:  10.1002/0471143030.cb1016s47
Online Posting Date:  June, 2010
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Abstract

The modulus of elasticity of the extracellular matrix (ECM), often referred to in a biological context as “stiffness,” naturally varies within the body, e.g., hard bones and soft tissue. Moreover, it has been found to have a profound effect on the behavior of anchorage‐dependent cells. The fabrication of matrix substrates with a defined modulus of elasticity can be a useful technique to study the interactions of cells with their biophysical microenvironment. Matrix substrates composed of polyacrylamide hydrogels have an easily quantifiable elasticity that can be changed by adjusting the relative concentrations of its monomer, acrylamide, and cross‐linker, bis‐acrylamide. In this unit, we detail a protocol for the fabrication of statically compliant and radial‐gradient polyacrylamide hydrogels, as well as the functionalization of these hydrogels with ECM proteins for cell culture. Included as well are suggestions to optimize this protocol to the choice of cell type or stiffness with a table of relative bis‐acrylamide and acrylamide concentrations and expected elasticity after polymerization. Curr. Protoc. Cell Biol. 47:10.16.1‐10.16.16. © 2010 by John Wiley & Sons, Inc.

Keywords: extracellular matrix; elasticity; hydrogel; gradient; polyacrylamide

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Fabrication of Statically Compliant Matrix Substrates of Varying Stiffness
  • Alternate Protocol 1: Fabrication of Radial‐Gradient Matrix Substrates of Varying Stiffness
  • Basic Protocol 2: Preparation of Matrix Protein Substrates of Varying Stiffness for Cell Culture
  • Support Protocol 1: Construction of a Gradient‐Gel Apparatus
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Fabrication of Statically Compliant Matrix Substrates of Varying Stiffness

  Materials
  • 0.1 M NaOH
  • Distilled H 2O
  • 3‐Aminopropyltriethoxysilane (APES)
  • 0.5% (v/v) glutaraldehyde in phosphate‐buffered saline (PBS; Cellgro, cat. no. 46‐013‐CM)
  • Dichlorodimethylsilane (DCDMS)
  • 40% (w/v) acrylamide stock solution (Sigma‐Aldrich, cat. no. A4058)
  • 2% (w/v) bis‐acrylamide stock solution (Sigma‐Aldrich, cat. no. M1533)
  • Phosphate‐buffered saline (PBS), optional
  • Tetramethylethylenediamine (TEMED)
  • 10% (wv) ammonium persulfate (APS)
  • 25‐mm circular coverslips (for 6‐well plate)
  • Hot plate
  • 35‐mm petri dish(es)
  • Kimwipes
  • 25 × 75–mm glass slides
  • Vacuum desiccator
  • Vortex mixer
  • 6‐well plate, optional

Alternate Protocol 1: Fabrication of Radial‐Gradient Matrix Substrates of Varying Stiffness

  Materials
  • 40% (w/v) acrylamide stock solution (Sigma‐Aldrich, cat. no. A4058))
  • 2% (w/v) bis‐acrylamide stock solution (Sigma‐Aldrich, cat. no. M1533)
  • Distilled water or phosphate‐buffered saline (PBS; Cellgro, cat. no. 46‐013‐CM)
  • Irgacure 2959 (Ciba)
  • Vacuum desiccator
  • Gradient gel apparatus
  • Photomask
  • 254‐nm UV light source
  • Chloro‐silane‐treated 25 × 75–mm glass slides ( protocol 1)
  • APES‐coated glass coverslips ( protocol 1)
  • 35‐mm petri dish(es) or 6‐well plates

Basic Protocol 2: Preparation of Matrix Protein Substrates of Varying Stiffness for Cell Culture

  Materials
  • PA hydrogels ( protocol 1 or protocol 2)
  • 0.2 mg/ml sufosuccinimidyl‐6‐(4′‐azido‐2′‐nitrophenylamino)‐hexanoate (sulfo‐SANPAH; Pierce Biotechnology)
  • 50 mM HEPES buffer, pH 8.5, filter sterilized
  • ECM protein(s) of choice
  • Distilled H 2O or phosphate‐buffered saline (PBS; Cellgro, cat. no. 46‐013‐CM), sterile
  • 320‐nm or 365‐nm UV light source
  • 37°C tissue culture incubator

Support Protocol 1: Construction of a Gradient‐Gel Apparatus

  Materials
  • Laser printer
  • Nitrocellulose film
  • Photomask design
  • 4 mil transparency film
  • Negative photomask design
  • 254‐nm UV light box
  • Spacers
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Figures

Videos

Literature Cited

Literature Cited
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