Here are presented several technologies advancing the state of microfluidic modeling of the vascular system through the integration of high throughput analysis equipment with microfluidic systems, allowing for the monitoring of cell-cell communication between the red blood cell (RBC) and endothelial cells. A review of the prior knowledge of the role of adenosine triphosphate (ATP) and nitric oxide (NO) release from the RBC is presented, along with the current understanding of the role these molecules play in vasodilation along with the endothelium. Here, this dissertation hypothesizes that hypoxic vasodilation of blood vessels requires ATP release from the RBC that stimulates NO synthesis in the endothelium, resulting in vasodilation. To investigate this hypothesis, a microfluidic model capable of quantitatively determining NO and culturing endothelial cells near a flowing RBC channel is fabricated. Techniques for fabrication of microfluidic devices are reviewed, along with detection and cell culture systems integrated in microfluidic systems. First presented is a novel system for analysis of NO released from RBCs under hypoxic conditions that uses a polydimethylsiloxane (PDMS) based microfluidic device incorporating a polycarbonate membrane. This membrane separates a flowing RBC sample from fluorogenic probe, DAF-FM, which when reacted with NO, has a larger fluorescence emission. This device was designed to be integrated into a fluorescence plate reader to obtain readout, which was a significant improvement over prior systems requiring custom detection platforms or microscopes for readout. Using this technology, a significant increase in NO release from RBCs under hypoxic conditions was observed.Next endothelial cell culture was incorporated onto the membrane of the device, and inhibition studies performed to investigate the origin of NO which reaches beyond an endothelial layer. In this model system, NO measured above an endothelial layer is representative of that available to the smooth muscle to induce vasodilation. Using this system, which integrated novel approaches of using the DAF-FM probe for NO in an extracellular manner and plate reader detection, it was shown that under hypoxic conditions, an increase in NO detected above the endothelium only is observed when RBC ATP release function and endothelial NO synthesis function is present, suggesting a pathway of hypoxic vasodilation requiring RBC ATP release and endothelial NO productionAlso, technology was developed enabling electrochemical oxygen detection within a flowing channel on a microfluidic device using epoxy-immobilized gold and silver wires as working and reference electrodes, respectively. This presents an easily reusable and low cost platform to potentially vary then detect the oxygen concentration in a flowing cell sample prior to other analysis. This would allow the investigation of hypoxic systems at oxygen concentrations other than completely oxygenated, or completely deoxygenated. Lastly, in efforts to explore other applications of the polycarbonate membrane based microfluidic devices, in particular their potential utility in drug screening and development. To this end, transport of several selected pharmaceutical molecules with the membrane based devices was investigated. In this process, it was discovered that some of the more hydrophilic molecules can absorb into PDMS based devices. To remove this limitation, techniques were developed which use the available fabrication equipment to produce PDMS devices to fabricate polystyrene devices. As polystyrene is frequently used in cell culture applications, this should allow for future work to more readily transfer cell culture protocols and techniques to microfluidic systems.
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