Date of Award
Doctor of Philosophy (PhD)
Biotechnology Science and Engineering
Debra M. Moriarity
Anoxemia., Cancer cells., Tumors.
Solid tumors can have areas of hypoxia. Hypoxia is associated with changes in tumor cell metabolism, with radiation therapy resistance, with chemotherapy resistance, and with tumor aggressiveness. Yet most cell culture research and drug studies are based on cell lines grown in atmospheric oxygen (normoxia). Our hypothesis was that differences in protein levels, differences in mitochondrial morphology, and differences in killing of cells by drug compounds in normoxic and hypoxic conditions would be observed. Normoxic conditions were achieved by culturing cell lines in humidified incubators supplemented with 5% CO2. Hypoxic conditions were achieved by culturing cells in a culture chamber, supplied by InQ Biosystems, which could be adjusted to 1.4% oxygen. The results of examining cells under both conditions showed that there is a difference in mitochondrial morphology in hypoxic versus normoxic cells and that the levels of many cellular proteins are different under hypoxia and normoxia. CA9 and HIF-1α protein levels are both increased in cancer cell lines cultured under hypoxic conditions. Nuclear ERα, which is high in normoxic cell culture conditions for estrogen receptor positive cells, was significantly decreased in these cells in hypoxic cultures. This information is important for treatment of cancers that are known to be estrogen dependent. Unfortunately the company supplying the hypoxia instrument, InQ Biosystems, went out of business so a different method had to be found to provide hypoxic cells for research. That led to the development of the gradient coverslip (gCVSL) as part of this research. The results for the gCVSL did compare favorably to the results of the InQ instrument, indicating that there were regions of hypoxia in gCVSL as expected. Thus, the gCVSL can achieve regions of hypoxia to mimic the tumor environment. The results demonstrate that the gCVSL method will provide a means of growing hypoxic cells for cancer research and for hypoxia drug studies. The gCVSL method is inexpensive, simple to perform, and readily available to all researchers.
Wright, Brenda Smith, "Gradient coverslip (gCVSL) : a novel way to culture cells under hypoxia" (2017). Dissertations. 120.