NEW MICROFLUIDIC SYSTEMS FOR SINGLE-CELL SIGNALING STUDIES IN IMMUNE CELLS
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Profiling immune cell signaling pathways requires techniques that allow for precise spatial cell positioning, control of the cellular microenvironment, and the ability to perform high-throughput multiplexed measurements at a single-cell resolution. Traditional tools often focus on bulk measurements that can mask the inherent cell heterogeneity or assays that enable a single snapshot of individual cells, therefore missing information on the temporal dynamics. Emerging microscale tools have been applied to improve on traditional approaches and also to introduce new ways of analyzing immune cell signaling events. This thesis presents microscale tools and image-based approaches for analyzing immune cell responses. The first application is designed for short-term (~ 1 hour) monitoring of calcium signaling in T cells upon stimulation with surface ligands. The platform enables high-density trapping of individual cells and monitoring of calcium dynamics with ~ 300 cells in a single field of view. I further developed a high-density cell trap array coupled with a dynamic soluble signal generator that enables temporal changes in the cellular microenvironment to be performed while simultaneously monitoring calcium changes in the cells. Building on these above mentioned platforms an integrated platform was developed to enable defined cell spatial positioning, stimulation with both surface-anchored molecules and soluble cues, monitoring of dynamic changes with fluorescence live imaging, incubation times of up to 24 hours and multiple assays to quantify changes in mRNA using single molecule Fluorescene In Situ Hybridization (smFISH) and protein expression by immunofluorescence staining. We further sought to advance the throughput at which the receptor/ligand interaction kinetics are measured in a more physiological context. I developed a microfluidic-based tool combined with automated operation and image processing that will allow for parallelization of the receptor/ligand interaction kinetics measurements. Finally, I utilize the microscale tools capabilities to develop an assay for separating leukocytes from less than 1 μL of whole blood. The platform enables leukocyte immunophenotyping from minute quantities of blood, and has potential applications when handling blood samples from small experimental animals like mouse, and for performing routine monitoring of a patient’s immune response. Overall, the systems developed in this study enable robust single-cell handling and easy fluid exchanges that facilitate the control of the cell microenvironment. In addition, the platforms developed are easy to use, and do not require sophisticated auxiliary equipment to function, making them easily transferrable to a typical biological laboratory.