Matrix remodeling in microfluidic co-culture

 描述（由申请人提供）：细胞外基质（ECM）的重塑是慢性呼吸道疾病（包括肺纤维化、肺动脉高压、哮喘和COPD）发病机制的核心。虽然已经将很多重点放在研究单个细胞类型以及它们如何与ECM相互作用上，但相对较少的注意力集中在ECM的稳态维持如何在多种肺驻留细胞类型之间协调，以及在损伤或疾病的情况下细胞相互作用如何恢复稳态或产生病理性基质重塑。阻止研究这些关键的细胞-细胞和细胞-基质相互作用的主要限制是缺乏实验上易于处理的多细胞培养系统，其包含能够长期重塑的3D基质。另一个主要的限制源于有限的数量，可用性，增殖能力，和原代人类细胞的表型稳定性，这是一个关键的资源，如果我们要阐明的“正常”稳态和“病理”基质重塑过程，人类健康和疾病的基础。我们的目标是开发一种细胞培养模型系统，其（1）促进在多种相互作用的原代肺细胞类型的控制下的稳态和病理性基质重塑的长期培养研究;（2）使得能够进行重复的非破坏性成像和取样以鉴定与基质重塑基础的细胞-细胞和细胞-基质相互作用相关并最终预测的细胞和可溶性线索;和（3）提供了研究少量原代人类细胞的平台，作为朝向增强的表型保真度和个性化医学的一步。这些设计目标将通过两个相互关联的具体目标来实现。在目标1中，我们将开发和优化微流体系统和培养条件，允许肺表皮/内皮和细胞外基质包埋的成纤维细胞稳定、长期共培养。我们将在共培养中评估ECM和组织重塑的多种生理指标，并使用已知促进体内基质/组织重塑的培养条件（例如TGF-β刺激）评估其灵敏度和信噪比。在目标2中，我们将把原代人类细胞纳入微流体共培养系统，作为迈向个性化医疗、治疗优先化和生物发现的一步。我们将使用已批准和失败的抗纤维化疗法来评估该系统的预测能力，作为概念验证。为了利用该系统的发现能力，我们将收集基质室流体样本进行蛋白质组学分析，以确定基质重塑过程中差异表达的新候选介质。该项目将产生一个新的实验平台，能够在基质重塑过程中重复实时分析细胞-细胞和细胞-基质相互作用，这是多种呼吸道疾病发病机制的核心过程。将低传代或无传代的原代人类细胞并入平台将增强实验的生物逼真度，并为肺基质重塑领域的个性化医学、治疗评估和生物发现提供独特的资源。

 DESCRIPTION (provided by applicant): Remodeling of the extracellular matrix (ECM) is central to the pathogenesis of chronic respiratory diseases, including pulmonary fibrosis, pulmonary hypertension, asthma and COPD. While much emphasis has been placed on studying individual cell types and how they interact with the ECM, comparatively little attention has focused on how homeostatic maintenance of the ECM is coordinated amongst multiple lung resident cell types, and how cell interactions in the setting of injury or disease either restore homeostasis or generate pathological matrix remodeling. A major limitation preventing investigation of these crucial cell-cell and cell-matrix interactions is the absence of experimentally tractable multicellular culture systems which incorporate 3D matrices capable of long-term remodeling. Another major limitation stems from the limited number, availability, proliferative capacity, and phenotypic stability of primary human cells, which are a critical resource if we are to elucidate the "normal" homeostatic and "pathological" matrix remodeling processes that underlie human health and disease. Our goal in this proposal is to develop a cell culture model system that (1) facilitates long-term culture study of homeostatic and pathologic matrix remodeling under the control of multiple interacting primary lung cell types; (2) enables repeated non-destructive imaging and sampling to identify cellular and soluble cues that correlate with, and ultimately predict, the cell-cell and cell-matrix interactions underlying matri remodeling; and (3) provides a platform for studying primary human cells in small quantities as a step toward enhanced phenotypic fidelity and personalized medicine. These design goals will be met through two interrelated specific aims. In Aim 1 we will develop and optimize a microfluidic system and culture conditions permitting stable, long-term co-culture of lung epi/endothelium and extracellular matrix-embedded fibroblasts. We will evaluate multiple physiologic metrics of ECM and tissue remodeling in co-culture, and evaluate their sensitivity and signal to noise ratio using culture conditions known to promote matrix/tissue remodeling in vivo (e.g. TGF-beta stimulation). In Aim 2 we will incorporate primary human cells into the microfluidic co-culture system as a step toward personalized medicine, therapeutic prioritization, and biologic discovery. We will use approved and failed anti-fibrosis therapies to assess the predictive capacity of this system as proof of concept. To capitalize on the discovery capabilities of the system, we will collect matrix compartment fluid samples for proteomic analysis to identify novel candidate mediators differentially expressed during matrix remodeling. This project will generate a new experimental platform that enables repeated real-time analysis of cell-cell and cell-matrix interactions during matrix remodeling, a process central to the pathogenesis of multiple respiratory diseases. Incorporation of primary human cells with low or no passaging into the platform will enhance the biofidelity of experiments and offer a unique resource for personalized medicine, therapeutic evaluation and biologic discovery in the realm of lung matrix remodeling.