3D Oxygen Microfluidic Platform for In Vitro Hypoxic Studies
Mauleon Ramos, Gerardo
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How cells and tissues respond at the microscale to a hypoxic insult is a fundamental question in angiogenesis as well as in stroke research. Due to the importance of oxygen in the cell’s viability and behavior, cell oxygen environment should be a critical factor in the experimental setting. Ironically, it is one of the most often overlooked components of cell culture experiments. There is a pressing need to create an in vitro platform that can recapitulate the oxygen environment experienced by cells in vivo. Towards this end, three different techniques were developed to aid in the study of hypoxic research in cells and tissues. In chapter 1, a microfluidic oxygenator is used to deliver constant oxygen to rodent brain slices, enabling the loading of the cell-permeant calcium indicator Fura-2/AM into cells of adult brain slices. When compared to traditional methods, the microfluidic oxygenator improves loading efficiency, measured by the number of loaded cells per unit area, for all tested age groups. Loading in slices from 1-year-old mice was achieved, which has not been possible with current bulk loading methods. This technique significantly expands the age range for which calcium studies are possible without cellular injection. In chapter 2, a 3D printed microfluidic oxygen mixer was designed to output desired oxygen levels for ease of experiment use. The unit requires two inputs of gasses and will output twenty-two different gas concentrations. When compared to other existing techniques, the 3D oxygen mixer provides simplicity, accuracy, and low expense. The proposed model can be used in hypoxia research where the microenvironment must be greatly controlled due to the extreme sensitivity of cells to oxygen levels. In chapter 3, a three dimensional environment, oxygen control, and advanced microfabrication techniques are brought together to create a 3D oxygen microfluidic platform where in vitro angiogenic hypoxic studies can be performed. Using this oxygen platform, the separate effect that growth factors and hypoxia have on the angiogenic response will be studied. Using this platform, angiogenesis was observed with no chemical factors being used as the driving force; instead, a hypoxic insult was used as the angiogenic factor.