Development of Photoreversible 4D Cell Culture Technologies

在历史上，细胞培养实验是生物学理解和药物候选筛选的基础 已经在平坦、坚硬的塑料衬底上进行了均匀的细胞群实验。越来越多的证据 这表明，这些僵硬的静态培养底物只会扩大体外和体内检测之间的差距。为 许多干细胞和多细胞构建物、2D底物无法概括组织结构 并引发与生物相关的反应。体内细胞周围的细胞外基质(ECM)是一种复合体， 与细胞交换生物物理和生化线索的异质和动态环境。ECM 力学和成分随时间的变化--在发育、再生、修复、疾病期间 进展和衰老--以及作为器官中三维位置的函数。以增加生理性的 与体外细胞培养实验相关，研究人员已经开发出生物和合成基质 这与组织的力学和水分含量大致相同。这些ECM模拟代表了一种妥协 在复杂性和可控性之间。我们将解决4D细胞培养的两个关键挑战：可逆的外部 对矩阵的控制，以及独立可调的刚度和应力松弛。 这项拟议的研究的目标是开发可调的4D细胞培养平台OptoGels 技术我们已经开发出粘弹性水凝胶，它可以通过两种不同的方式变硬和变软 可见光的波长。通过在动态共价键附近安装可逆光开关偶氮苯 通过硼酸酯的键合，我们可以用光来控制硼酸酯形成的结合常数。在预赛中 实验中，我们实现了高达9kpa的光调刚度，僵硬的时空图案化， 和细胞相容性。拟议研究的具体目标是：(1)确定设计参数 将其作为细胞培养材料的多功能性最大化，(2)基准间充质干细胞和 光凝胶中上皮细胞的机械生物学对照文献数据和商业材料，以及(3)工程师 空间控制的光凝胶促进多能干细胞来源的功能成熟 肝细胞。可与现有材料进行比较的功能读数和指标包括YAP 核移位、干细胞分化、细胞扩散、细胞迁移、基因表达和酶 活动。这项研究的预期产品是一套具有机械功能的光凝胶配方 可以在生理上相关的长度和时间范围内进行调整的属性。这样做的长期目标是 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.