Fabrication of Hydrogels with Steep Stiffness Gradients for Studying Cell Mechanical Response

Abstract

Many fundamental cell processes, such as angiogenesis, neurogenesis and cancer metastasis, are thought to be modulated by extracellular matrix stiffness. Thus, the availability of matrix substrates having well-defined stiffness profiles can be of great importance in biophysical studies of cell-substrate interaction. Here, we present a method to fabricate biocompatible hydrogels with a well defined and linear stiffness gradient. This method, involving the photopolymerization of films by progressively uncovering an acrylamide/bis-acrylamide solution initially covered with an opaque mask, can be easily implemented with common lab equipment. It produces linear stiffness gradients of at least 115 kPa/mm, extending from ∼1 kPa to 240 kPa (in units of Young's modulus). Hydrogels with less steep gradients and narrower stiffness ranges can easily be produced. The hydrogels can be covalently functionalized with uniform coatings of proteins that promote cell adhesion. Cell spreading on these hydrogels linearly correlates with hydrogel stiffness, indicating that this technique effectively modifies the mechanical environment of living cells. This technique provides a simple approach that produces steeper gradients, wider rigidity ranges, and more accurate profiles than current methods.

Biomaterials for Enhancing Neuronal Repair

“…Polyacrylamide can be used to create hydrogels with gradient stiffness ranging from ∼1 to 240 kPa. Polyacrylamide can act as a strong analog to the endogenous ECM when invested with proteins and chemical signals specific to the cell of interest (Sunyer et al, 2012;Lee et al, 2015b). Hydrogels can be constructed in planar or 3D configurations maintaining precise control over the elastic modulus (Chatterjee et al, 2011;Wylie et al, 2011).…”

Regulation of Endothelial Cell Adherence and Elastic Modulus by Substrate Stiffness

“…Parameter s indicates the Poisson's ratio and was set to 0.5 due to the assumption of incompressibility for hydrogels (Sunyer et al 2012). A previously published algorithm (Guo & Akhremitchev 2006;Nikkhah et al 2011) was used to obtain the young's modulus of substrates based on d parameter in Hertz's model.…”

UV Dose Governs UV-Polymerized Polyacrylamide Hydrogel Modulus

“…Although TEMEDpolymerized gels offer a range of elastic moduli (Figure 1), there are several distinct advantages to using UV polymerization. First, it allows for the formation of gradient gels which are excellent platforms for the investigation of cell motility, durotaxis, or differentiation [1,10,12]. Second, UV polymerization also allows for gel and surface patterning, providing a tool for evaluating cell behavior as a function of surface topography or composition [18,19].…”

“…Photocrosslinking with various photoinitiators, such as Irgacure 2959, has been more recently employed for the fabrication of PAA hydrogels with a stiffness gradient [1,10,12], or for the quick preparation of large PAA hydrogel arrays for applications such as drug screening [8]. Photocrosslinking circumvents the use of toxic catalysts and is typically much faster-on the order of 1-5 min [8].…”