Stiffness Measurement of Soft Silicone Substrates for Mechanobiology Studies Using a Widefield Fluorescence Microscope

Summary

Substrates with stiffness in the kilopascal-range are useful to study the response of cells to physiologically relevant micro-environment stiffness. Using just a widefield fluorescence microscope, the Young's modulus of soft silicone gels can be determined using an indentation with a suitable sphere.

Abstract

Soft tissues in the human body typically have stiffness in the kilopascal (kPa) range. Accordingly, silicone and hydrogel flexible substrates have been proven to be useful substrates for culturing cells in a physical microenvironment that partially mimics in vivo conditions. Here, we present a simple protocol for characterizing the Young's moduli of isotropic linear elastic substrates typically used for mechanobiology studies. The protocol consists of preparing a soft silicone substrate on a Petri dish or stiff silicone, coating the top surface of the silicone substrate with fluorescent beads, using a millimeter-scale sphere to indent the top surface (by gravity), imaging the fluorescent beads on the indented silicone surface using a fluorescence microscope, and analyzing the resultant images to calculate the Young's modulus of the silicone substrate. Coupling the substrate's top surface with a moduli extracellular matrix protein (in addition to the fluorescent beads) allows the silicone substrate to be readily used for cell plating and subsequent studies using traction force microscopy experiments. The use of stiff silicone, instead of a Petri dish, as the base of the soft silicone, enables the use of mechanobiology studies involving external stretch. A specific advantage of this protocol is that a widefield fluorescence microscope, which is commonly available in many labs, is the major equipment necessary for this procedure. We demonstrate this protocol by measuring the Young's modulus of soft silicone substrates of different elastic moduli.

Introduction

Cells in soft tissues reside in a micro-environment whose stiffness is in the kilopascal range¹, in contrast to tissue culture dishes whose stiffness is several orders of magnitude higher. Early experiments with cells on extracellular matrix protein-coated soft substrates showed that the substrate stiffness influences how cells move on as well as adhere to the extracellular matrix beneath^2,3. In fact, the substrate stiffness fundamentally influences the cell function⁴ in a manner similar to pervasive biochemical signals. Polyacrylamide gels (coated with extr....

Protocol

1. Fabrication of Soft Silicone Substrate

1. Weigh out 1.75 g of the A component and 1.75 g of the B component (A:B = 1:1) from the soft silicone elastomer kit using (polystyrene) weighing trays.
2. Add the A component to the B component in the weighing tray and mix them together for 5 min using an appropriate applicator stick.
3. Add the above mixture to a 35 mm Petri dish. Allow the mixture to spread evenly across the Petri dish for a couple of minutes.
   Note: The choice of the Petri dish diameter and the amount of soft silicone will determine the soft silicone thickness. Here, the thickness will be around 3.5 mm; more about choosing the e....

Results

Using the protocol detailed above, we prepared soft silicone in a 35 mm Petri dish, cured it at 70 °C for 30 min and coupled fluorescent microbeads (and collagen I) to the top surface as schematically depicted in Figure 1. Deep UV has been used previously for the eventual protein coupling to substrates¹³. Note that (I) the curing conditions used here are specific to this soft silicone and (II) the indentation measurement is perform.......

Discussion

While the sphere indentation method is easy to implement, careful attention must be paid to the choice of indentor and the thickness of the soft silicone sample. The equation used to calculate the Young's modulus is valid under a set of conditions¹¹and these are typically satisfied when the thickness of the silicone sample is > 10% of the indentor radius and < ~13x the indentor radius. We found that a silicone thickness of 5 - 10x the indentor radius was a good choice, wherein the samp.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
CY 52-276 A/B silicone elastomer kit	Dow Corning	CY 52-276	Store at room temperature
Thermo Scientific Pierce EDC	Fisher Scientific	PI22980	Store at -20°C
Thermo Scientific Pierce Sulfo-NHS crosslinker	Fisher Scientific	PI-24510	Store at 4°C
Carboxyl fluorescent pink particles, 0.4-0.6 µm, 2 mL	Spherotech, Inc.	CFP-0558-2	Store at 4°C, do not freeze
1.0 mm Acid washed Zirconium beads	OPS Diagnostics LLC	BAWZ 1000-250-33
Deep UV chamber with ozone evacuator	Novascan Technologies, Inc.	PSD-UV4, OES-1000D
Wide field fluorescence microscope	Leica Microsystems	DMi8
Collagen I, from rat tail	Corning	354236	Stock concentration = 4 mg/ml; store at 4°C
ImageJ-NIH	N/A	N/A	public-domain software