The Extracellular vesicles (EVs) secreted by cells play a crucial role in intercellular communication by transporting chemical signaling molecules like proteins, lipids, DNA, and RNA through the extracellular environment. The importance of EVs in signaling pathways has only recently been widely recognized, and while many studies investigate EV secretion in bulk cell samples, few studies are published on this occurrence at a single cell level. By examining single cell-derived EVs, variations in the cell populations could be better characterized, demonstrating cellular heterogeneity in populations of cells. Cellular heterogeneity affects the behavior of individual cells in complex cellular networks, which is why novel techniques for single cell isolation are being developed to better characterize individual cell phenotypes. One specific method of sample purification is droplet microfluidics, in which the sample is contained by aqueous droplets suspended in an oil layer that can be manipulated using microfluidics. Benefits of this method include low reagent volume requirements and high throughput.

In my thesis, an experimental workflow for the isolation of single NK cell-derived EVs is presented and optimized. NK cells are cultured, dyed, attached to magnetic beads, encapsulated in an oil droplet at a single cell level, and inputted into the “Coalesce-Attract-Resegment Wash” (CAR-Wash) system developed by the Bailey lab. Using microfluidic junctions and magnetic forces, the CAR-Wash separates NK cell-bead complexes from any other cells or waste in the solution. The droplets are then observed over time to monitor EV secretion. Aspects such as encapsulated cell viability, generation of fluorescent exosomes, and blocking of non-specific binding between EVs and paramagnetic beads are investigated through extensive testing. This is done using techniques such as nanoparticle tracking analysis and fluorescence microscopy. To apply the EV isolation approach described above, NK cells are exposed to IL-18 and their EV secretion is monitored to determine how IL-18 influences EV biogenesis on a single cell level.

DATE: Thursday, April 14, 2022
TIME: 3:00 PM
Zoom: https://umich.zoom.us/j/93165855971 (passcode: bmedefense)
Chair: Prof. Sunitha Nagrath