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

描述（由申请人提供）：本研究的总体目标是开发一种新型微流体装置，用于表征白细胞在生理上真实的微环境中与内皮的相互作用（滚动、粘附和迁移）。白细胞在对由物理、化学或生物刺激引起的组织损伤/感染的早期反应中起关键作用。由于白细胞-内皮细胞相互作用的重要性，已经开发了几种体外模型来研究白细胞粘附级联的不同方面。流动室已被开发用于研究滚动和粘附现象，Boyden/transwell室已被用于迁移研究。然而，所使用的流动室过于简化，缺乏微环境的尺度和几何形状，并且不能模拟轮回。类似地，transwell/Boyden室不考虑体内观察到的流体剪切和尺寸/拓扑，白细胞迁移的终点测量是半定量的，不提供白细胞迁移的实时可视化，并且是劳动密集型的。由于没有模型可以在单一测定中表征粘附和迁移，因此对粘附级联的理解和抗炎药物的开发受到阻碍。例如，可以在Boyden室中停止迁移的药物可能不会影响流动存在下的滚动/粘附，反之亦然。为了克服这些局限性，我们建议开发和展示一种新的微流控装置，用于表征白细胞粘附级联。与目前的体外模型相比，该器械将解决并促进单个系统中单个步骤的直接评估，包括白细胞进入血管外组织空间的滚动、牢固停滞（粘附）、扩散和外渗。本项目的具体目标是：1）开发一种新型的微流体装置（MFD），模拟白细胞粘附/迁移级联，2）使用粘附/迁移级联中特定步骤的阻断剂/抑制剂证明这种微流体装置的独特性和效率，3）在小鼠模型中使用活体显微镜观察MFD。这种新型的微流体系统不仅使我们能够在真正模拟微血管环境的解剖学现实模型中研究白细胞-组织相互作用，而且还将为各种治疗领域的先进药物发现和递送研究提供试验平台。一个多学科的学术和工业研究人员团队，具有微循环和细胞粘附，微加工/微流体，计算建模和活体显微镜的专业知识，将开发和验证这种独特的白细胞滚动，粘附和迁移的体外模型。 公共卫生相关性：炎症反应是从哮喘和动脉粥样硬化到炎症性肠病的许多病理状况的基础。我们计划开发一种新型的微流体装置，用于表征白细胞与内皮细胞的相互作用，模拟体内条件。这将是第一个微流控系统，将允许在单个系统中直接评估和观察白细胞进入血管外组织空间的滚动、牢固停滞（粘附）、扩散和外渗。

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.