Mammalian cell culture has gained importance in biotechnology for the development of therapeutic and diagnostic agents. Among them, Chinese hamster ovary (CHO) cells are regarded as the mammalian cell "workhorse". The use of CHO cell line for the production of recombinant proteins used in human therapy has reached a level of industrial production. However, a major problem encountered in in vitro cultures is cell death via apoptosis. Since apoptosis leads to the loss of viability of mammalian cells in vitro, especially in serum-free media. This is important and necessary to prevent the activation of apoptosis cascade and increase their cell viability and enhance their cellular robustness. The overall goal of this study is to improve our understanding of the cellular and physiological determinants of apoptosis and its relationship with other cellular functions. Apoptosis is a result of a very complex network of signaling pathways triggered from both inside and outside of the cell and a highly regulated pathway by both pro-apoptotic and anti-apoptotic proteins that promote cell survival or cell death. Although many causes of apoptotic process in mammalian cell cultures had been researched in the past and have been discussed in recent years, a lot need to be explored. In order to bring novel strategies to understand apoptosis in mammalian cell cultures, our study was not only focused on the apoptotic pathway but also expand to metabolic network to set up a link between cell growth and apoptosis. In our project, we applied systems biology methods in a mammalian cell line (CHO TF 70R), to understand the relationship between cellular metabolism and apoptosis in a typical serum free culture medium. After establishing the basic culture platform, the effects of culture conditions on initiating apoptosis will be evaluated. Healthy and apoptotic cell samples were identified and isolated using Fluorescence Activated Cell Sorting (FACS) and Magnetic Activated Cell Sorting (MACS), respectively. A comprehensive study of CHO cellular metabolism was made using a metabolic flux network to compare and analyze by metabolic flux analysis (MFA) to get more information on cell metabolism and apoptotic behavior. Furthermore, 2-NBDG combined with Annexin V-PE was also successfully applied to estimate the glucose uptake rate in real early apoptotic cells. In summary, we used the integration of the data generated by MFA to understand apoptotic behavior and establish a correlation between cell regulation and apoptosis. It will help us to identify the changes during the onset of apoptosis process will be studied by using proteomics tools to analyze the protein up-regulation or down-regulation in different cell status in the future.
| Identifer | oai:union.ndltd.org:RICE/oai:scholarship.rice.edu:1911/70459 |
| Date | January 2011 |
| Contributors | Gonzalez, Ramon |
| Source Sets | Rice University |
| Language | English |
| Detected Language | English |
| Type | Thesis, Text |
| Format | 155 p., application/pdf |
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