Spatiotemporal Regulated Click Hydrogels for 3D Cell Culture

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The aim of this proposal is to fully develop click chemistry reactions to create a dynamic in vitro 3D cell culture platform that will enable researchers to explore how cell-material interactions influence important cellular functions. These biomaterials will give the user control of specific physical, chemical, and biological cues comprising the cell microenvironment in both time and space. To accomplish this significant challenge, the proposed work will exploit the classic alkyne/azide click chemistry, modified to enable a cytocompatible copper-free polymerization, to synthesize basic hydrogels as a simplified mimic of the extracellular matrix in the presence of cells. The biochemical functionality of the gels will then be tuned using a new variant of click chemistry, a thiol-ene photoaddition reaction, that is fully compatible with peptide chemistry and allows spatiotemporally regulated introduction of biological epitopes. Finally, a photodegradable linker will be integrated into the crosslinks of the base gel formulation to allow light directed degradation of the material. Using these three independent and cytocompatible reactions for gelation, photodegradation, and photocoupling, dynamic biomaterials systems will be created that will provide newfound opportunities to probe the dynamic exchange of information between cells and their microenvironment. The specific aims of this work are to: (1) synthesize click-based hydrogels using orthogonal chemistries that allow manipulation of the material properties through photodegradation and introduction of biological epitopes through a photocoupling reaction, (2) manipulate the properties of the above hydrogels through photodegradation, photocoupling, and combined photodegradation and photocoupling, (3) culture human mesenchymal cells (hMSCs) on 2D gel surfaces and characterize morphology, cytoskeletal organization, and focal adhesions as a function of elasticity and patterned adhesive ligand presentation, (4) encapsulate hMSCs in 3D and examine their response to material-directed morphological changes and spatially varying adhesive ligand distribution, (5) train a diverse group of undergraduate and graduate students at the interface of polymer chemistry and molecular biology, and (6) disseminate these results to the industrial and academic public to assure maximum impact on fundamental and applied biomaterials chemistry.BROADER IMPACTS: This new class of multifunctional hydrogels will help overcome numerous problems with existing 3D cell culture platforms by providing a facile means for manipulating material functionality and mechanics with spatial and temporal control. These new materials will enable researchers to probe fundamental biological questions via a variety of previously unattainable 3D cell studies. The efficient nature of the gel chemistry will allow its adoption by a wide array of non-experts. These material systems should find applications for basic 3D cell culture, design of tissue engineering matrices, platforms for drug delivery and screening, stand-alone biomaterials implants, as well as other non-biological applications. The multidisciplinary team environment in the Anseth laboratory, coupled with long-standing collaborations with world-class clinical and biological laboratories, will provide an exceptional educational environment for multiple graduate and undergraduate students. The PI and her research group have a history of extensive outreach to high school students and the general public, and the team will work diligently through multiple mechanisms to highlight the impact of biomaterial science in benefiting society.