Spatiotemporal Regulated Click Hydrogels for 3D Cell Culture

该提案的目的是充分开发点击化学反应，以创建一个动态的体外3D细胞培养平台，使研究人员能够探索细胞-物质相互作用如何影响重要的细胞功能。这些生物材料将使用户能够控制特定的物理、化学和生物线索，包括时间和空间上的细胞微环境。为了完成这一重大挑战，提出的工作将利用经典的炔/叠氮化物点击化学，修改为使细胞兼容的无铜聚合，在细胞存在的情况下合成基本的水凝胶，作为细胞外基质的简化模拟物。然后，凝胶的生化功能将使用一种新的点击化学变体，即巯基光加成反应进行调整，该反应与肽化学完全兼容，并允许在时空上调节生物表位的引入。最后，一种可光降解的连接剂将被整合到基础凝胶配方的交联中，以允许材料的光定向降解。利用这三种独立的细胞相容性反应进行凝胶化、光降解和光耦合，动态生物材料系统将被创建，这将为探索细胞及其微环境之间的动态信息交换提供新的机会。这项工作的具体目标是：(1)利用正交化学方法合成基于点击的水凝胶，通过光降解和光偶联反应引入生物表位来操纵材料性质；(2)通过光降解、光偶联以及光降解和光偶联的组合来操纵上述水凝胶的性质；(3)在二维凝胶表面培养人间充质细胞（hMSCs）并表征形态、细胞骨架组织。(4)在3D中封装hMSCs，并研究它们对材料导向的形态变化和空间变化的粘附配体分布的响应；(5)在聚合物化学和分子生物学的界面上培养多样化的本科生和研究生群体；(6)向工业界和学术界传播这些结果，以确保对基础和应用生物材料化学产生最大的影响。更广泛的影响：这种新型多功能水凝胶将有助于克服现有3D细胞培养平台的许多问题，通过空间和时间控制提供一种简便的方法来操纵材料功能和力学。这些新材料将使研究人员能够通过各种以前无法实现的3D细胞研究来探索基本的生物学问题。凝胶化学的高效性质将允许它被广泛的非专家采用。这些材料系统应该在基本的3D细胞培养、组织工程基质设计、药物输送和筛选平台、独立生物材料植入以及其他非生物应用中找到应用。Anseth实验室的多学科团队环境，加上与世界一流的临床和生物实验室的长期合作，将为多名研究生和本科生提供卓越的教育环境。PI和她的研究小组有着广泛接触高中生和公众的历史，团队将通过多种机制努力工作，突出生物材料科学在造福社会方面的影响。

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.