The impact of mechanical stresses induced by flow and stretch on barrier function in airway epithelial cells

背景：许多已知的关于呼吸道上皮的基本生物学知识都是从静态的体外细胞培养系统中推断出来的。因此，我们对呼吸道上皮细胞生物学的了解可能是有限的，并且没有意识到动态力对上皮屏障功能中重要的生物学特性的影响。我们的首要目标是将流动和拉伸的生理范围纳入我们的体外呼吸道上皮细胞培养模型，并探索屏障功能特性。 在体外细胞培养系统中加入FLOW已经表明，基线粘液分泌比先前认为的要高得多。此外，压缩应力的加入表明，呼吸道上皮细胞在单层内运动，并不固定在位置上。将Flow和Stretch结合到呼吸道上皮细胞的体外细胞培养模型中，可能会揭示未知的生物学。 目的：使用体外细胞培养模型来探索i)生理范围的流动，ii)生理范围的拉伸，以及iii)流动和拉伸的组合对呼吸道上皮细胞屏障功能的影响 方法：所有三个AIMS都进行了实验，通过跨皮细胞电阻(TEER)、分子转运、伤口修复和基因表达来探索上皮屏障功能。 我们将使用一种人类呼吸道上皮细胞系CALU-3来进行我们的实验，因为这种细胞系产生了严格的机械屏障，并已被验证可用于分子运输研究。实验将在水下单层培养的细胞上进行，如果可能的话，还会在气液界面培养条件下进行。我们将把我们的细胞培养与聚二甲基硅氧烷(PDMS)芯片上器官装置相结合，以实现灌流和拉伸的一体化。对于灌流实验，将使用注射器泵将细胞暴露于基于体内静息和峰值流速的一定范围的流速。对于拉伸实验，将使用带有闭塞阀的改装注射器泵系统来诱导压力变化，这些压力变化在体内转换为对拉伸的响应。为了探索流动和拉伸之间相互作用的可能性，我们将在我们的PDMS设备上组合这些机械力。 意义：其他人的初步工作和我们的初步数据表明，由流动和拉伸引起的机械力影响呼吸道上皮细胞生物学。我们拟议的研究计划将通过整合目前缺乏传统细胞培养系统的生理条件，探索呼吸道上皮细胞生物学的未知领域，为未来的基础和健康研究发现创造知识基础。此外，我们的计划将开发和验证受知识产权保护的技术和方法，这些技术和方法可以适用于其他类型的细胞，以研究组织特定的机械力如何影响功能。

Background: Much of the basic biology known about the airway epithelium has been deduced from static in vitro cell culture systems. Our understanding of airway epithelial cell biology may therefore be limited and under appreciate the impact of dynamic forces on biological properties important in epithelial barrier function. Our overarching objective is to incorporate physiological ranges of flow and stretch into our in vitro airway epithelial cell culture models and explore barrier function properties. Incorporation of flow into in vitro cell culture systems has showed that baseline mucus secretion is much higher than previously considered. Furthermore, incorporation of compressive stress showed that airway epithelial cells move within a monolayer and are not fixed in position. Incorporating flow and stretch into in vitro cell culture models of airway epithelial cells is likely to reveal unexplored biology. Aims: Use in vitro cell culture models to explore the impact of i) a physiological range of flows, ii) a physiological range of stretch, and iii) a combination of flow and stretch on airway epithelial cell barrier function Methods: All three aims have experiments with outcome measurements that explore epithelial barrier function measured by transepithelial electrical resistance (TEER), molecular transport, wound repair, and gene expression. We will use a human airway epithelial cell line, Calu-3, to perform our experiments as this cell line creates tight mechanical barriers and has been validated for molecular transport studies. Experiments will be performed on cells grown under submerged monolayer and, where possible, air-liquid interface culture conditions. We will combine our cell cultures with a polydimethylsiloxane (PDMS) organ-on-a-chip device to allow integration of perfusion and stretch. For perfusion experiments, a syringe pump will be used to expose cells to a range of flow rates based on in vivo resting and peak flow rates. For stretch experiments, an adapted syringe pump system with an occlusion valve will be used to induce pressure changes that are transduced in vivo in response to stretch. To explore the potential for interactions between flow and stretch, we will combine these mechanical forces on our PDMS device. Significance: Nascent work by others and our preliminary data suggest that mechanical forces induced by flow and stretch impact airway epithelial cell biology. Our proposed research program will explore unknown areas of airway epithelial cell biology by integrating physiological conditions currently absent from conventional cell culture systems, creating a foundation of knowledge for future basic and health research discoveries. Furthermore, our program will develop and validate technology and methods, protected by intellectual property, that can be adapted to other cell types to study how tissue specific mechanical forces impact function.