Development and Validation of Migration Assay for Cellular Backpacks Adhered to Human and Murine Monocytes for Targeted Drug-Delivery

Abstract

The Mitragotri Lab has been developing immune cell-based therapies to improve drug targeting and accumulation, particularly to cross the blood-brain barrier for the treatment of traumatic brain injury. We use cellular ‘backpacks’ that attach to the surface of immune cells and traffic in a membrane-bound state to sites of inflammation for delivery of cytokines, small molecules, and other payloads that modulate the immune microenvironment. This thesis optimizes and validates a transwell migration assay to quantify the migration of monocytes and monocyte-backpack units through an human umbilical vein endothelial cell (HUVEC) monolayer. Optimized parameters include 5 μm transwell membrane insert pore size, 10 ng/mL MCP1 chemokine gradient in lower media transwell chamber, 200k cells/well HUVEC and monocyte/monocyte-backpack seeding densities, and a 24 hour incubation period after seeding of monocytes in the upper transwell chamber. This system was validated using both murine and human primary monocytes. Preliminary results show that adhesion of backpacks to monocytes does not significantly interfere with the ability of monocytes to migrate across an endothelial layer, further validating the potential use of backpacks for targeted drug delivery.