Laser-based techniques for manipulating the single-cell environment

Abstract

The environment encountered by a single cell in vivo is a complex and dynamic system that is often simplified experimentally via ex vivo and in vitro methods. As our understanding of cell response in these basic environments grows, there is a corresponding need for techniques that modify traditional cell culture in ways that better mimic the complexities of in vivo systems. This dissertation examines how the three dimensional (3D) properties of a focused pulsed laser can be incorporated within existing techniques to dynamically manipulate these microenvironments in the presence of single cells. As a modification on existing microfluidic technology for chemically dosing cells, it is shown how a cost-effective microchip laser can be used to ablate microscopic pores in a thin, biocompatible polymer membrane. These pores serve as conduits for introducing dosing reagents in close proximity to cultured cells combining subcellular resolution with spatial and temporal control. Because reagent flow is physically separated from the cell-culture flow chamber by this polymer membrane, the geometry of the reagent flow cell can be altered to accommodate multiple reagents flowing in parallel with minimal mixing due to the laminar flow characteristics of microfluidic devices. By manipulating reagent flow, a single cell can be dosed at opposing ends by distinct reagents or by defined, stable gradients of a single reagent. Additionally, these dosing streams can be switched with subsecond temporal resolution or dynamically mixed to study potential synergistic or antagonistic effects. To define the physical environment surrounding small populations of cells, an existing platform for mask-directed multiphoton lithography is used to create biocompatible protein-based microstructures for studying cancer-cell migration and invasion in physically confined regions. In these studies, a variety of 3D shapes incorporating spatial gradients are examined with invasive cell types. Additionally, these methods have been modified to allow for in situ fabrication of gelatin microstructures with 3D resolution around suspended somatic cells by covalently binding a photosensitizing molecule to the protein prior to fabrication. The architecture of these microstructures is designed to provide a variety of 3D confinement scenarios with biological relevance.