Perfused 3D Tissue Surrogates for Complex Cell-Cell Communication Systems

描述（由申请人提供）：现有的培养和动物模型和方法之间存在着巨大的差距，这些模型和方法可以提取细胞-细胞通信网络的详细信息，这些信息控制着系统对扰动的反应，例如炎症线索。网络本质上以一种复杂的、相互关联的方式运作，并且经常在网络的特定点上表现出非直觉的干预结果，正如许多靶向治疗在临床环境中失败所证明的那样，在目前可用的临床前试验中取得了有希望的结果。细胞-细胞通讯网络包括细胞释放到细胞外环境的因子（如细胞因子、蛋白酶）以及细胞内信号。虽然大量的努力都集中在简单细胞培养系统中通过单个刺激信号直接处理产生的细胞内信号上，但尚不清楚这些信号与不同细胞类型作为动态级联在顺序时间点产生的多种信号相互作用产生的信号之间的相关性。阐明多种群细胞系统中细胞外因子相互作用的重要方面对于理解组织病理生理学至关重要，但在体内或传统细胞培养系统中进行研究非常困难。在这个项目中，我们将开发变革性的新方法，将细胞-细胞通信网络的实时分子探针和随之而来的细胞行为整合到复杂的生理3D培养中，允许从这些培养中获得多路复用的动态信息，以响应系统变量的特定操作，包括所涉及的细胞群和外部扰动，如炎症线索。我们的目标是建立原始人类系统的模型，以作为体内复杂性的接近模拟，因此我们专注于开发不依赖于细胞群体遗传操作的新方法来生成有关系统运行的信息。我们的整体项目将通过三个并行但相互交织的努力来推进：开发连接细胞外和细胞内网络的通信模式的分析形式，并提供一个框架，用于从细胞外介质的测量（如蛋白质组学分析）中识别关键的细胞外节点；开发新的生物材料微环境，以多路复用的方式控制和记录细胞周围环境中局部细胞通信信号的关键节点，具有高时空分辨率；并将这些方法整合到微尺度灌注培养系统中，通过控制细胞外基质特性、培养几何形状、局部氧张力和机械应力等因素，培养适当的细胞和组织生理学。我们工作中的一项重大创新是以协同方式将这些方法联系起来，以提供可广泛用于各种组织系统的系统，并应用于一系列个体疾病，包括那些性别二态反应突出的疾病。

DESCRIPTION (provided by applicant): A tremendous gap exists between available culture and animal models and methods that can extract detailed information on cell-cell communication networks governing system responses to perturbations, such as an inflammatory cue. Networks inherently operate in a complex, interlinked fashion, and often exhibit non-intuitive outcomes from intervention at a particular point in the network, as evidenced by failure of many targeted therapeutics to operate in the clinical setting after promising results in currently- available preclinical trials. Cell-cell communication networks comprise factors the cells release into the extracellular milieu (e.g., cytokines, proteases) along with intracellular signals. While an immense amount of effort has focused on intracellular signals generated in simple cell culture systems by straightforward treatment with individual stimulatory cues, it is not clear how relevant those are to the signals arising from interplay of multiple cues being produced at sequential time-points by diverse cell types as dynamic cascades. Elucidating vital aspects of the interplay of extracellular factors in multi-population cellular systems is crucial for understanding tissue pathophysiology but is exceedingly difficult to study in vivo or in traditional cell culture systems. In this project, we will develop transformative new methods to integrate real time molecular probes of cell-cell communication networks and consequent cell behavior into complex, physiological 3D cultures, allowing multiplexed, dynamic information to be derived from these cultures in response to specific manipulations of the system variables, including cell populations involved and external perturbations such as inflammatory cues. Our goal is to build models of primary human systems to serve as close mimics of in vivo complexity, hence we focus on developing new methods that do not rely on genetic manipulation of the cell populations to generate information about systems operation. Our overall project will advance via three parallel but interwoven efforts: development of an analytical formalism for communication modes that connects extracellular and intracellular networks and provides a framework for identifying key extracellular nodes from measurements (such as proteomic analysis) of extracellular medium; development of new biomaterials microenvironments that both control and record key nodes in local cell communication signals in the pericellular environment in a multiplexed manner, with high spatial and temporal resolution; and integration of these approaches into microscale perfused culture systems that foster appropriate cellular and tissue physiology through control of factors including extracellular matrix properties, culture geometry, local oxygen tension, and mechanical stresses. A major innovation in our work is linking these approaches in a synergistic manner to provide systems that can be used broadly in a wide variety of tissue systems with application to an array of individual diseases, including those where sexually dimorphic responses are prominent. PUBLIC HEALTH RELEVANCE: The goal of this project is transform our ability to probe cell-cell communication networks in human cell systems via linking systems biology with tissue engineering. Our overall project will advance via three parallel but interwoven efforts: development of an analytical formalism for communication modes that connects extracellular and intracellular networks and provides a framework for identifying key extracellular nodes from measurements (such as proteomic analysis) of extracellular medium; development of new biomaterials microenvironments that both control and record key nodes in local cell communication signals in the pericellular environment in a multiplexed manner, with high spatial and temporal resolution; and integration of these approaches into microscale perfused culture systems that foster appropriate cellular and tissue physiology through control of factors extracellular matrix properties, culture geometry, local oxygen tension, and mechanical stresses.