Matrix remodeling in microfluidic co-culture

微流控共培养中的基质重塑

• 批准号: 9087443
• 负责人: Daniel J. Tschumperlin
• 金额: $ 20.92万
• 依托单位: MAYO CLINIC ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-04-11 至 2018-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9087443
• 关键词: Asthma; Attention; Biological Models; Biomedical Engineering; Blood capillaries; Cell Communication; Cell Count; Cell Culture Techniques; Cells; Chronic; Chronic Obstructive Airway Disease; Coculture Techniques; Cues; Development; Disease; Endothelium; Epithelium; Extracellular Matrix; Fibroblasts; Fibrosis; Gel; Goals; Health; Homeostasis; Human; Image; Image Analysis; Individual; Injury; Investigation; Liquid substance; Lung; Lung diseases; Maintenance; Mediator of activation protein; Microfluidics; Nature; Noise; Paracrine Communication; Pathogenesis; Pathologic; Perfusion; Physiological; Process; Proteomics; Pulmonary Fibrosis; Pulmonary Hypertension; Resources; Sampling; Side; Signal Transduction; System; Therapeutic; Time; Tissues; Transforming Growth Factor beta; capillary; cell type; design; differential expression; in vivo; meetings; novel; personalized medicine; prevent; public health relevance; research study; stem; therapeutic evaluation

 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.

 描述（由申请人提供）：细胞外基质（ECM）的重塑对于慢性呼吸系统疾病的发病机制至关重要，包括肺纤维化、肺动脉高压、哮喘和慢性阻塞性肺病（COPD），而研究个体细胞类型以及它们如何发挥作用已成为人们关注的重点。与 ECM 相互作用，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，以及损伤或疾病情况下细胞相互作用如何恢复稳态或产生病理基质对这些关键的细胞-细胞和细胞-基质相互作用的研究的一个主要限制是缺乏实验上可处理的多细胞培养系统，其中包含能够长期重塑的3D基质，另一个主要限制源于数量、可用性和增殖性的限制。如果我们要阐明人类健康和疾病的“正常”稳态和“病理性”基质重塑过程，那么原代人类细胞的能力和表型稳定性是至关重要的资源。建议开发一种细胞培养模型系统，该系统（1）促进在多种相互作用的原代肺细胞类型的控制下进行稳态和病理基质重塑的长期培养研究；（2）能够进行重复的无损成像和采样以识别细胞与基质重塑相关的细胞-细胞和细胞-基质相互作用相关并最终预测的可溶性线索；(3) 提供了一个用于少量研究原代人类细胞的平台，作为增强表型保真度和个性化医疗的一步。这些设计我们将通过两个相互关联的具体目标来实现目标 1，我们将开发和优化微流体系统和培养条件，以实现肺外皮/内皮细胞和细胞外基质嵌入成纤维细胞的稳定、长期共培养。共培养中 ECM 和组织重塑的指标，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）评估其敏感性和信噪比。目标 2，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、治疗优先顺序和生物发现的一步。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，作为证明。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，以识别基质重塑过程中差异表达的新型候选介质。该项目将生成一个新的实验平台，能够对细胞进行重复实时分析。 -细胞和基质重塑过程中的细胞-基质相互作用是多种呼吸系统疾病发病机制的核心过程，将原代人类细胞掺入平台中的量很少或没有，将增强实验的生物保真度，并为个性化医疗、治疗评估和治疗提供独特的资源。肺基质重塑领域的生物学发现。