Matrix remodeling in microfluidic co-culture

 描述(申请人提供)：细胞外基质(ECM)重塑是慢性呼吸系统疾病发病机制的核心，包括肺纤维化、肺高压、哮喘和COPD。虽然对单个细胞类型以及它们如何与ECM相互作用的研究受到了很大的重视，但相对较少的关注是如何在多种肺驻留细胞类型之间协调ECM的动态平衡维持，以及在损伤或疾病的背景下细胞相互作用如何恢复动态平衡或产生病理性基质重塑。阻碍研究这些关键的细胞-细胞和细胞-基质相互作用的一个主要限制是缺乏实验上容易处理的多细胞培养系统，该系统结合了能够长期重塑的3D基质。另一个主要限制来自于人类原代细胞数量、可获得性、增殖能力和表型稳定性的有限，如果我们要阐明构成人类健康和疾病的“正常”内稳态和“病理性”基质重塑过程，这些细胞是一个关键资源。我们在这个建议中的目标是开发一种细胞培养模型系统，该系统(1)在多种相互作用的原代肺细胞类型的控制下促进对动态平衡和病理基质重塑的长期培养研究；(2)使重复的非破坏性成像和采样能够识别与细胞-细胞和细胞-基质相互作用相关的细胞和可溶性线索，并最终预测细胞-细胞和细胞-基质重塑；以及(3)为少量研究原代人类细胞提供一个平台，作为迈向增强表型保真度和个性化药物的一步。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控系统和培养条件，使肺上皮/内皮细胞和细胞外基质包埋的成纤维细胞能够长期稳定地共培养。我们将评估共培养中细胞外基质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(例如转化生长因子-β刺激)来评估它们的敏感性和信噪比。在目标2中，我们将把原代人类细胞合并到微流控共培养系统中，作为走向个性化医学、治疗优先和生物发现的一步。我们将使用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，我们将收集基质间隔液样本进行蛋白质组分析，以确定在基质重塑过程中差异表达的新的候选介体。该项目将产生一个新的实验平台，能够重复实时分析基质重塑过程中的细胞-细胞和细胞-基质相互作用，这是多种呼吸系统疾病发病机制的核心过程。将低传代或不传代的原代人类细胞纳入平台将提高实验的生物保真度，并为肺基质重塑领域的个性化药物、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.