Phototunable Click-based Hydrogels for 3D Cell Culture: Dynamic Biochemical and Biomechanical Tailorability of the Cell Niche

DeForest, Cole Alexander

Graduate Thesis Or Dissertation

Phototunable Click-based Hydrogels for 3D Cell Culture: Dynamic Biochemical and Biomechanical Tailorability of the Cell Niche Public Deposited

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Citeable URL: https://scholar.colorado.edu/concern/graduate_thesis_or_dissertations/p5547r56q

Abstract

To better understand how cells receive and respond to signals presented by their external surroundings, polymer-based hydrogels have emerged as highly tailorable constructs for assaying cell function in well-defined microenvironments where the effects of user-dictated functionalities can be individually probed. This thesis research aimed to develop synthetic strategies to create and modify 3D cell culture platforms that permit researchers to explore in real time how cell-material interactions influence important biological function. Specifically, cytocompatible hydrogels were formed via a novel strain-promoted azide-alkyne cycloaddition reaction between a tetrafunctional poly(ethylene glycol) and a difunctional enzymatically-degradable peptide sequence, which enabled cell-mediated active remodeling of the local material. By varying the monomer composition during material formulation, hydrogels of different initial physical properties (e.g., moduli, swelling ratio, crosslinking density) were created in the presence of encapsulated primary cells and established cell lines with high viability. Subsequently, we introduced a thiol-ene reaction as a means to alter the chemical makeup of the hydrogel post-gelation. By photoinitiating this reaction, precise spatiotemporal control over the network's biochemical functionalization was demonstrated, and synthetic peptides were patterned into the substrate to visualize enzymatic activity locally and direct 3D cell spreading within user-defined subvolumes of the gels. Additionally, a photodegradable o-nitrobenzyl ether moiety was introduced within the peptide precursors that enabled chemical crosslinks within the network to be photocleaved and for the material physical properties to be controlled in time and space. By establishing light conditions that permitted both photoreactions to be performed independently, gels were created that enabled user-defined manipulation of material biophysical and biochemical properties that were used to direct cell motility in 3D. Finally, these chemistries were exploited to introduce reversibly biomolecules to the synthetic culture platform, and experiments demonstrate the ability to promote cell adhesion to defined regions on a hydrogel surface, along with subsequent release of the captured cells, through dynamic presentation of the RGDS epitope. Collectively, this thesis research utilized multiple orthogonal reactions for the formation of hydrogel biomaterials whose physical and chemical properties were subsequently modified in time and space to probe and direct basic cellular functions (e.g., adhesion, motility).

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