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.