Development of Photoreversible 4D Cell Culture Technologies

从历史上看，细胞培养实验是生物学理解和候选药物筛选的基础 已经在平坦、坚硬的塑料基底上用均匀的细胞群进行了。越来越多的证据 表明这些坚硬的、静态的培养基质只会扩大体外和体内测定之间的差距。为 许多干细胞和多细胞构建体，2D基质无法重现组织结构 并引出生物学上相关的反应。体内细胞周围的细胞外基质（ECM）是一种复合物， 异质和动态的环境，与细胞交换生物物理和生物化学线索。ECM 在发育、再生、修复、疾病等过程中， 进展和衰老--以及作为器官中三维位置的函数。为了提高生理 在体外细胞培养实验的相关性，研究人员已经开发出生物和合成基质 模拟组织的力学和含水量。这些ECM模拟物代表了一种妥协 复杂性和控制之间的关系我们将解决4D细胞培养的两个关键挑战： 控制矩阵，并独立可调的刚度和应力松弛。 拟议研究的目标是开发OptoGels，这是一种用于4D细胞培养的可调平台 技术.我们已经开发了粘弹性水凝胶，可以通过两种不同的方法硬化和软化， 可见光的波长。通过安装一个可逆的光开关，偶氮苯，邻近动态共价键， 硼酸酯键，我们可以控制与光形成硼酸酯的结合常数。初步 实验中，我们已经实现了高达9千帕的光可调刚度，刚度的时空图案， 和细胞相容性。所提出的研究的具体目的是（1）建立设计参数， OptoGels，以最大限度地发挥其作为细胞培养材料的多功能性，（2）基准间充质干细胞和 OptoGels中的上皮细胞机械生物学，对照文献数据和商业材料，和（3）工程师 增强多能干细胞衍生的功能成熟的空间控制的OptoGels 肝细胞可以与已建立的材料进行比较的功能读数和指标包括雅普 核转位、干细胞分化、细胞铺展、细胞迁移、基因表达和酶 活动这项研究的预期产品是一套OptoGel配方， 这些特性可以在生理相关的长度和时间尺度上进行调整。长期目标是 研究是开发用户定义的细胞培养材料，使生物医学研究人员能够理解和 控制与人类健康相关的细胞过程。

Historically, the cell culture experiments underlying biological understanding and drug candidate screening have been performed with homogeneous populations of cells on flat, stiff plastic substrates. Mounting evidence suggests that these stiff, static culture substrates only widen the gap between in vitro and in vivo assays. For many stem cells and multicellular constructs, 2D substrates are unable to recapitulate the tissue architecture and elicit biologically relevant responses. The extracellular matrix (ECM) surrounding cells in vivo is a complex, heterogeneous, and dynamic environment that exchanges biophysical and biochemical cues with cells. ECM mechanics and composition change as a function of time—during development, regeneration, repair, disease progression, and aging—and as a function of 3-dimensional location in organs. To increase the physiological relevance of in vitro cell culture experiments, researchers have developed biological and synthetic matrices that approximate the mechanics and water content of tissue. These ECM mimics represent a compromise between complexity and control. We will address two key challenges for 4D cell culture: reversible external control over the matrix, and independently tunable stiffness and stress relaxation. The objective of the proposed research is to develop OptoGels, a tunable platform for 4D cell culture technology. We have developed viscoelastic hydrogels that can be stiffened and softened by two different wavelengths of visible light. By installing a reversible photoswitch, azobenzene, adjacent to a dynamic covalent boronic ester linkage, we can control the binding constant for boronic ester formation with light. In preliminary experiments, we have achieved phototunable stiffnesses up to 9 kPa, spatiotemporal patterning of stiffness, and cytocompatibility. The Specific Aims of the proposed research are to (1) establish design parameters for OptoGels to maximize their versatility as cell culture materials, (2) benchmark mesenchymal stem cell and epithelial cell mechanobiology in OptoGels against literature data and commercial materials, and (3) engineer spatially controlled OptoGels that enhance the functional maturation of pluripotent stem cell-derived hepatocytes. Functional readouts and metrics that can be compared to established materials include YAP nuclear translocation, stem cell differentiation, cell spreading, cell migration, gene expression, and enzyme activity. The anticipated products of this research are a suite of OptoGel formulations with mechanical properties that can be tuned over physiologically relevant length and time scales. The long-term goal of this research is to develop user-defined cell culture materials that allow biomedical researchers to understand and control cellular processes relevant to human health.