Metabolic flux analysis of mammalian cell metabolism using multiple isotopic tracers and mass spectrometry
| Author(s) | Ahn, Woo Suk | |
| Date Accessioned | 2022-09-13T17:22:20Z | |
| Date Available | 2022-09-13T17:22:20Z | |
| Publication Date | 2012 | |
| SWORD Update | 2022-08-11T19:08:10Z | |
| Abstract | Metabolic flux analysis (MFA) is a powerful technology to characterize intracellular metabolism in living cells using isotopic tracers and mass spectrometry. Therefore, in the past two decades, MFA techniques have been developed to study biological systems. However, the applications of MFA for mammalian cells have been limited due to the complexity of cellular metabolism even though mammalian cells are key platforms for biopharmaceutical production and biomedical research. Here, we present two applications for glycolysis and gluconeogenesis systems. First, we describe the analysis of metabolic fluxes in CHO metabolism at fed-batch mode. We established two metabolic models of CHO cells for non-stationary and stationary <super>13</super>C-MFA. It was found that cellular metabolism in CHO cells were significantly rewired from exponential growth to stationary phases during culture. The results provide a solid foundation for applications such as cell line development and medium optimization. Second, we describe gluconeogenesis metabolism of Fao rat hepatoma cells perturbed by transcription factors. Using multiple isotopic tracers and combined <super>13</super>C-MFA, we observed the regulations of metabolic fluxes by transcriptional activators and inhibitors for gluconeogenesis metabolism. The discovery and the applied MFA techniques can allow us to evaluate the pharmaceutical drug for metabolic disease, e.g. Type II diabetes. And finally, we provide the comprehensible procedures to be considered for <super>13</super>C-MFA technique: isotopic and metabolic stationarity, isotopic tracer design, key measurements, multiple isotopic tracers and model validation. | en_US |
| Advisor | Antoniewicz, Maciek R. | |
| Degree | Ph.D. | |
| Department | University of Delaware, Department of Chemical and Biomolecular Engineering | |
| Unique Identifier | 1344343790 | |
| URL | https://udspace.udel.edu/handle/19716/31349 | |
| Language | en | |
| Publisher | University of Delaware | en_US |
| URI | https://login.udel.idm.oclc.org/login?url=https://www.proquest.com/dissertations-theses/metabolic-flux-analysis-mammalian-cell-metabolism/docview/2704864962/se-2?accountid=10457 | |
| Keywords | Isotopic tracers | |
| Keywords | Mammalian cells | |
| Keywords | Mass spectrometry | |
| Keywords | Metabolic engineering | |
| Keywords | Metabolic flux analysis | |
| Keywords | System biology | |
| Title | Metabolic flux analysis of mammalian cell metabolism using multiple isotopic tracers and mass spectrometry | en_US |
| Type | Thesis | en_US |