Development of effective CHO-K1 host system targeting at nutrient-regulated insulin-like growth factor I (IGF-I) pathway /
An effective mammalian cell culture host system expressing therapeutic proteins is a combination of cell line's inherent characteristics with efficient use of nutrients that is able to provide desirable output of high cell viability. Insulin-like Growth Factor I (IGF-I) has been shown to have t...
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| Main Author: | |
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| Format: | Thesis |
| Language: | English |
| Published: |
Kuala Lumpur :
Kulliyyah of Engineering, International islamic University Malaysia,
2014
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| Subjects: | |
| Online Access: | http://studentrepo.iium.edu.my/handle/123456789/4617 |
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| Summary: | An effective mammalian cell culture host system expressing therapeutic proteins is a combination of cell line's inherent characteristics with efficient use of nutrients that is able to provide desirable output of high cell viability. Insulin-like Growth Factor I (IGF-I) has been shown to have the ability to promote cell proliferation, while also having complex interactions with other constituents in the media. Therefore, it is hypothesized that if IGF-I pathway is effectively manipulated it could lead to achieving the desired high cell density culture. This present study is designed to develop a CHO-K1 based host system by understanding (IGF-I) gene and protein expression in this cell line and its expression relationship between constituents of media. It is confirmed that both IGF-I gene and protein are expressed in CHO-K1 cells, through reverse transcriptase real-time PCR analysis and enzyme-linked immunosorbent assay (ELISA) respectively. Using a three level Full Factorial Design, the optimal media composition of 10 % (v/v) serum and 0.500 mM glutamine was found to contribute to high cell density of 8.870 X 105 cells/ml in T-flask. The optimal media composition was validated and gave 12.500 x 105 cells/ml; an increase of 26.936 % from culturing in standard formulation of 10 % (v/v) serum and 2 mM glutamine. The culture with optimal media then reached 23.300 x 105 cells/ml (46.352 % increased) when scaled-up in 500 ml spinner vessel. The culture also reached higher cell density (16.600 x 105 cells/ml); increase of 24.699 % from 12.500 x 105 cells/ml) when adapted in zero glutamine. Results from Full Factorial Design showed that the quadratic term of glutamine plays an important role for high cell density. This also supports the observation that the cells reached high cell concentration when cultured in zero glutamine media. Based on multivariate data analysis (MVDA) using Principal Component Analysis (PCA) and Partial Least Square Discriminant Analysis (PLS-DA), the high cell density achieved in low glutamine was found to be correlated with high expression of IGF-I gene and protein. This may be governed through growth hormone signaling and IGF-I signaling pathway as shown in the pathway analysis performed in this work. In conclusion, the study showed that the IGF-I pathway which is known for its role in cell proliferation is responsive to the regulation by nutrients. The relationship between the reduced requirement of glutamine with the high expression of IGF-I gene and protein further improves the efficiency of the system. The host system (CHO-K1 cell line and the improved media formulation) obtained from this study can now serve as a platform for producing bio-products of interest |
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| Physical Description: | xxiv, 334 leaves : ill. ; 30cm. |
| Bibliography: | Includes bibliographical references (leaves 210-219). |