CHARACTERIZATION AND APPLICATION OF MICROFLUIDIC DEVICES FOR STUDYING CELLULAR MIGRATION AND CHEMOTAXIS
Abstract
Microfluidic devices, often called "lab on a chip", have been utilized in the fields of physics, chemistry, and biology. In this thesis, the application of a microfluidic device as an in vitro assay to study cellular responses to biochemical and biophysical stimuli with a particular focus on the migration and invasion of cancer cells is discussed. The device consists of an array of well pairs joined by 10 microchannels. With device miniaturization and efficient use of space, a large number of experiments can therefore be performed simultaneously on a single platform. Polydimethylsiloxane (PDMS), an optically transparent silicone rubber, was used to fabricate the device and permitted direct visualization of cellular dynamics during migration with the aid of a phase contrast inverted microscope. Using finite element analysis, gradient formation simulations were conducted and showed that chemical gradients could be rapidly generated in zero-flow conditions and remained stable for several days without additional maintenance. This approach allowed for comparatively rapid and simple device analysis without the need for lengthy and resource-intensive fabrication processes. Several microchannel configurations were modeled to examine the steady-state gradient profile, and it was found that linear and nonlinear concentration gradients could be passively generated and maintained for extended durations. The utility of the device as a platform for studying chemotaxis was demonstrated using several different cell lines. Lung and breast carcinoma cells were found to migrate rapidly through the microchannels of the device towards a gradient of epidermal growth factor, a potent chemoattractant. Prostate cancer cells were exposed to gradients of soluble factors present in various organs, and it was found that the cells migrated preferentially to bone, brain, and testes lysates, Immunocytochemical analysis revealed that cells that entered the channels exhibited upregulation of vimentin, a mesenchymal marker. The role of the novel gene Migration and Invasion Enhancer 1 (MIEN1) in promoting cell migration in breast cancer was also investigated. Overexpression of MIEN1 was associated with increased migration. It was also found that phosphorylation of the canonical immunoreceptor tyrosine-based activation motif (ITAM) is critical to the function of MIEN1. The possible role of MIEN1 in mediating collective migration through the promotion of cell-cell adhesion was also investigated, and although MIEN1-overexpressing cells displayed an increased tendency to form aggregates, immunostaining for several classic cell-cell adhesion molecules revealed no difference between the two conditions. With this platform, multiple cell lines and experimental conditions can be implemented and monitored simultaneously. The microfluidic device can be used to study both chemotaxis and innate migratory potential of adherent cells. Furthermore, in situ molecular interrogation of cells by means of immunocytochemistry is demonstrated. With minimal modifications, this platform could be used to study a variety of cell migration-based phenomena.