A novel microfluidic device for the study of leukocyte adhesion and migration

描述(申请人提供)：本研究的总体目标是开发一种新的微流控设备，用于在生理上真实的微环境中表征白细胞与内皮细胞的相互作用(滚动、黏附和迁移)。白细胞在对物理、化学或生物刺激引起的组织损伤/感染的早期反应中起着关键作用。由于白细胞与内皮细胞相互作用的重要性，已发展了几种体外模型来研究白细胞黏附级联反应的不同方面。流动室被用来研究滚动和粘着现象，Boyden/Transwell室被用来研究迁移。然而，所使用的流室过于简化，缺乏微环境的规模和几何形状，并且无法模拟轮回。同样，Transwell/Boyden小室不考虑体内观察到的液体剪切和大小/拓扑，白细胞迁移的终点测量是半定量的，不能提供白细胞迁移的实时可视化，并且是劳动密集型的。由于没有一种模型可以同时描述黏附和迁移的特征，因此对黏附级联的理解和抗炎药物的开发受到了阻碍。例如，一种可以在Boyden小室中阻止迁移的药物可能不会在有流动的情况下影响滚动/粘连，反之亦然。为了克服这些局限性，我们建议开发并展示一种用于表征白细胞粘附级联的新型微流控装置。与目前的体外模型相比，该设备将在单个系统中解析和促进对单个步骤的直接评估，包括滚动、牢固地阻止(粘连)、扩散和外渗白细胞进入血管外组织空间。本项目的具体目标是1)开发一种模拟白细胞黏附/迁移级联的新型微流控装置(MFD)，2)使用黏附/迁移级联中特定步骤的阻滞剂/抑制剂来展示这种微流控装置的独特性和效率，3)在小鼠模型中使用活体显微镜验证MFD。这一新颖的微流控系统不仅使我们能够在解剖学上真实模拟微血管环境的模型中研究白细胞与组织的相互作用，而且还将为各种治疗领域的先进药物发现和输送研究提供一个试验台。一个由学术和工业研究人员组成的多学科团队，拥有微循环和细胞黏附、微制造/微流体、计算建模和活体显微镜方面的专业知识，将开发和验证这一独特的白细胞滚动、黏附和迁移的体外模型。 与公共卫生相关：炎症反应是许多病理情况的基础，从哮喘和动脉粥样硬化到炎症性肠病。我们计划开发一种新的微流控设备来表征白细胞与内皮细胞的相互作用，以模拟体内的条件。这将是第一个允许直接评估和观察单个系统中白细胞滚动、牢固阻止(粘连)、扩散和渗入血管外组织空间的微流控系统。

DESCRIPTION (provided by applicant): The overall objective of this study is to develop a novel microfluidic device for characterizing leukocyte interactions with the endothelium (rolling, adhesion, and migration) in physiologically realistic microenvironments. Leukocytes play a key role in the early response to tissue injury/infection resulting from physical, chemical or biological stimuli. Due to the significance of the leukocyte-endothelium interactions, several in vitro models have been developed to study different aspects of the leukocyte adhesion cascade. Flow chambers have been developed to study rolling and adhesion phenomena, and Boyden/transwell chambers have been used for migration studies. However, the flow chambers used are oversimplified, lack the scale and geometry of the microenvironment and cannot model transmigration. Similarly, transwell/Boyden chambers do not account for fluid shear and size/topology observed in vivo, the end point measurement of leukocyte migration is semi-quantitative, do not provide real-time visualization of leukocyte migration, and are labor intensive. Since there are no models that can characterize both adhesion and migration in a single assay, the understanding of the adhesion cascade and the development of anti-inflammation drugs has been hindered. For example, a drug that can stop migration in Boyden chambers may not influence rolling/adhesion in the presence of flow and vice-versa. To overcome these limitations, we propose to develop and demonstrate a novel microfluidic device for characterization of the leukocyte adhesion cascade. In contrast with current in vitro models, this device will resolve and facilitate direct assessment of individual steps including rolling, firm arrest (adhesion), spreading and extravasations of the leukocytes into the extra-vascular tissue space in a single system. The specific aims of this project are to 1) Develop a novel microfluidic device (MFD) that mimics the leukocyte adhesion/migration cascade, 2) Demonstrate uniqueness and efficiency of this microfluidic device using blockers/suppressors of specific steps in the adhesion/migration cascade, 3) Validate the MFD using intravital microscopy in a mouse model. This novel microfluidic system will not only enable us to study leukocyte-tissue interactions in anatomically realistic models that truly mimic the microvascular environment, but also will provide a test bed for studies of advanced drug discovery and delivery in a variety of therapeutic areas. A multidisciplinary team of academic and industrial researchers with expertise in microcirculation and cell adhesion, microfabrication/microfluidics, computational modeling, and intravital microscopy will develop and validate this unique in vitro model of leukocyte rolling, adhesion and migration. PUBLIC HEALTH RELEVANCE: The inflammatory response is the basis for a number of pathological conditions ranging from asthma and atherosclerosis to inflammatory bowel disease. We plan to develop a novel microfluidic device for characterizing leukocyte interactions with the endothelium that mimics the in vivo condition. This will be the first microfluidic system that will allow for direct assessment and observation of rolling, firm arrest (adhesion), spreading and extravasations of leukocytes into the extra-vascular tissue space in a single system.