Astley, Kelly
(2010).
The co-modulation of apoptosis and the cell cycle.
University of Birmingham.
Ph.D.
Abstract
In modern times we have become increasingly reliant on mammalian cell culture for the production of biopharmaceuticals; therefore research aimed at improving the characteristics of the cell-lines being used for recombinant protein production is essential. In this study I have examined the hypothesis that the creation of a CHO cell-line in which the expression of p21 \(^C\)\(^I\)\(^P\)\(^1\)and Bcl-2 could be combined would un-couple cell growth from cellular proliferation resulting in a significant increase in both the rate of production and culture viability. Analysis of key metabolites together with changes in cell volume, total protein and mitochondrial activity indicate that following the initiation of p21\(^C\)\(^I\)\(^P\)\(^1\)-expression cells undergo an increase in their protein synthesis machinery and that the energy, previously required for cell division may be diverted towards cell growth and product formation. In addition to the requirement of cell-lines with high production capacities, the biopharmaceutical industry is under constant pressure to develop growth media able to facilitate high yields without the need for the addition of protein or serum. This means it is often necessary to adapt high producing cell-lines to growth in such a defined chemical environment, a process which has proven to be both extremely long and costly. In this thesis I have successfully developed a method for the swift adaptation of commercially important cell-lines to growth within a chemically defined bio-processing environment. I have shown that the expression of p21\(^C\)\(^I\)\(^P\)\(^1\)is able to reduce the need for extracellular growth factors and that by combining the expression of p21\(^C\)\(^I\)\(^P\)\(^1\) and Bcl-2 it is possible to further reduce the time required for successful adaptation, supporting the well established theory that Bcl-2 plays an important role in apoptotic signaling pathways.
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