Abstract
We here present a micropatterning strategy to introduce small molecules and ligands on patterns of arbitrary shapes on the surface of poly(acrylamide)-based hydrogels. The main advantages of the presented approach are the ease of use, the lack of need to prefabricate photomasks, the use of mild UV light and biocompatible bioconjugation chemistries, and the capacity to pattern low-molecular-weight ligands, such as peptides, peptidomimetics, or DNA fragments. To achieve the above, a monomer containing a caged amine (NVOC group) was co-polymerized in the hydrogel network; upon UV light illumination using a commercially available setup, primary amines were locally deprotected and served as reactive groups for further functionalization. Cell patterning on various cell adhesive ligands was demonstrated, with cells responding to a combination of pattern shape and substrate elasticity. The approach is compatible with standard traction force microscopy (TFM) experimentation and can further be extended to reference-free TFM.
Highlights
Understanding how cells sense and respond to the physical and mechanical properties of their insoluble microenvironment, i.e., their extracellular matrix (ECM), is a major challenge of mechanobiology research
The amplitude and dynamics of these forces depend on substrate viscoelasticity, and in turn determine the tension and force loading rate experienced by mechanosensing proteins present at focal adhesions (FAs).[1]
An unmet challenge for the majority of existing patterning methods for pAAm hydrogels is the patterned immobilization of short peptide or peptidomimetic ligands. Techniques to pattern such low-molecular-weight ligands have been reported for other hydrogel systems;[23,44,45] here, we demonstrated as a proof of principle, the patterning in lines of integrin peptide or peptidomimetic ligands on pAAm hydrogels, and visualized cell adhesion using optical microscopy
Summary
Understanding how cells sense and respond to the physical and mechanical properties of their insoluble microenvironment, i.e., their extracellular matrix (ECM), is a major challenge of mechanobiology research. The above results demonstrate how control over the size of adhesive patterns on soft elastic hydrogels can be used to study cell behavior and open the way for studying the combinatorial effects of substrate stiffness, cell shape, and ligand type. The technical limitations of the current setup (PRIMO system with 20× objective) allowed patterning of 1 μm circular patterns, with an interspot distance of 3 μm (Figures 6C and S4) These gels were functionalized with fibronectin and sulfo-SANPAH prior to seeding a rat embryonic fibroblast cell line (REF52) that stably express paxillin-coupled to yellow fluorescent protein (REFYFP‐PAX). An analogous analysis as for the standard TFM substrates was performed (Figure 6D,E and Movie S3), demonstrating the applicability of this approach to measure substrate deformations
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