Studying the Effects of Matrix Stiffness on Cellular Function using Acrylamide-based Hydrogels

Summary

The effect of substrata stiffness on cellular function can be modeled in vitro using polyacrylamide hydrogels of varying compliances.

Abstract

Tissue stiffness is an important determinant of cellular function, and changes in tissue stiffness are commonly associated with fibrosis, cancer and cardiovascular disease^1-11. Traditional cell biological approaches to studying cellular function involve culturing cells on a rigid substratum (plastic dishes or glass coverslips) which cannot account for the effect of an elastic ECM or the variations in ECM stiffness between tissues. To model in vivo tissue compliance conditions in vitro, we and others use ECM-coated hydrogels. In our laboratory, the hydrogels are based on polyacrylamide which can mimic the range of tissue compliances seen biologically¹². "Reactive" cover slips are generated by incubation with NaOH followed by addition of 3-APTMS. Glutaraldehyde is used to cross-link the 3-APTMS and the polyacrylamide gel. A solution of acrylamide (AC), bis-acrylamide (Bis-AC) and ammonium persulfate is used for the polymerization of the hydrogel. N-hydroxysuccinimide (NHS) is incorporated into the AC solution to crosslink ECM protein to the hydrogel. Following polymerization of the hydrogel, the gel surface is coated with an ECM protein of choice such as fibronectin, vitronectin, collagen, etc.

The stiffness of a hydrogel can be determined by rheology or atomic force microscopy (AFM) and adjusted by varying the percentage of AC and/or bis-AC in the solution¹². In this manner, substratum stiffness can be matched to the stiffness of biological tissues which can also be quantified using rheology or AFM. Cells can then be seeded on these hydrogels and cultured based upon the experimental conditions required. Imaging of the cells and their recovery for molecular analysis is straightforward. For this article, we define soft substrata as those having elastic moduli (E) <3000 Pascal and stiff substrata/tissues as those with E >20,000 Pascal.

Protocol

Preparation

• Coverslips should be autoclaved.
• Sterile distilled or deionized water should be used to prepare solutions and for washing coverslips.
• AC (40% w/v) and bis-AC (1% w/v) solutions are sterilized by 0.2 μm filtration. Prepare 10% ammonium persulfate (APS; 100μg/ml water) shortly before use and sterile filter. Replace the APS solution monthly.
• Chemical reagents such as 3-APTMS, chloroform, glutaradehyde, NHS, and SurfaSil that cannot be autoclaved are kept in an assigned bottle used solely for the preparation of hydrogels.
• For best results, hydrogels should be utilized within a couple of days after the overnig....

Discussion

A crucial element of the hydrogel polymerization process is to avoid air bubble formation which will allow cells to bind to the glass coverslip rather than the ECM-coated hydrogel itself. This can be prevented by carefully pipetting the polymerization solution after vortexing and visually making sure that no air bubbles have become trapped in the gel. We always recommend preparing additional "reactive" coverslips and hydrogels to ensure having enough for experimentation.

Particular attention .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Glutaraldehyde, 70%	Sigma-Aldrich	G7776	Store at -20°C
3-APTMS (3-Aminopropyltrimethosysilane 97%)	Sigma-Aldrich	281778	Store at room temperature
SurfaSil Siliconizing Fluid	Thermo Fisher Scientific, Inc.	42800	Store at room temperature
NHS (N-hydroxysucinimide Ester)	Sigma-Aldrich	A-8060	Store at 4°C Replace monthly
Albumin, bovine serum, essentially fatty acid free	Sigma-Aldrich	A6003-100G	Store at 4°C
Coverslips (25mm)	Fisher Scientific	12-545-86 25 Cir 1D
Coverslips (18mm)	Fisher Scientific	12-545-84 18 Cir 1D