High throughput microfluidic technologies for cell separation and single-cell analysis

Abstract

The heterogeneity of individual cellular behavior in response to physical and chemical stimuli has raised increasing attention in many biological processes. There is great incentive in developing techniques for high throughput single-cell measurements and manipulations. Particularly, cell size has been recognized as an important parameter in single cell study and pericellular protease activity plays a key role in regulating the microenvironment of individual cells. Therefore, this thesis focuses on establishing new methods to address the issues of cell size and single cell protease measurement. We first develop a size-based cell separation technique using Dean-coupled inertial microfluidic sorter. Separation of cells by size before downstream assays might be beneficial in simplifying the system and facilitating the discovery of rare subpopulations through enrichment of cells with certain size range or cell cycle phase. By investigating the particle focusing and separation mechanisms in curved microfluidic channel, we develop a novel design of inertial microfluidic sorter with higher separation resolution and then demonstrate its capacity in leukocyte isolation from blood. This novel cell sorter would be a promising alternative to many other cell separation problems. We then establish a microfluidic platform for functional measurement of single cell pericellular proteases, including both those secreted and expressed on cell surface. We apply the platform to studying the PMA-mediated protease response of HepG2 cells at single-cell level and reveal the diversity in the dynamic patterns of single-cell protease activity profile upon drug stimulation. We also present the preliminary exploration of single-cell protease activity behavior in anticancer drug resistance development. Lastly, we explore the applicability of our platform for single-cell shedding measurement. Protease-mediated molecular shedding is one of the key mechanisms through which individual cells actively regulate their own microenvironment. However, the amount of molecules being shed for individual cells is extremely low, posing significant challenges in detecting shedding quantitatively. By means of analytical analysis and numerical simulations, we investigate the intrinsic noise of low-abundance molecule detection. Experimental characterizations have also been performed to evaluate the impact of practical factors on actual readout variation.