Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.577424
Title: Adaptation to suspension growth : analysing the surface of suspension growth adapted Chinese hamster ovary cells
Author: Walther, Christa G.
Awarding Body: University of Sheffield
Current Institution: University of Sheffield
Date of Award: 2013
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Abstract:
Many bio-pharmaceutical production processes are based upon the use of mammalian cell lines, such as Chinese hamster ovary (CHO) cells, capable of proliferation as single cells in suspension in a synthetic environment. Routine use of CHO cells as production vehicles requires a lengthy “adaptation” process from the wild-type adherent clone to clones capable of proliferation in a suspension environment depleted of exogenous growth factors and cell-matrix contacts. Different approaches have been applied in this study to gain a better understanding of the changes on the cell surface occurring as a response to changes in their environment, comparing four cell lines (CCL61, AML, S cells and CHO-S) adapted to suspension or adherence growth condition. Biochemistry and mass spectrometry methods showed differences in surface protein composition for the cell lines. The comparison of the expression of cell-to-cell adhesion molecules revealed a highly variable bimodal distribution on S cells which was not seen on CCL61. Analysis of the expression level of integrins, the main interaction partner of serum components, indicated that integrin expression is not generally down-regulated on suspension-adapted CHO cells. The integrin conformation on the cell surface, analysed by confocal microscopy, revealed a specific conformation, especially with regard to integrin beta 1, characterised by an even, net-like distribution of integrin clusters over the surface of the cells. This specific integrin conformation, which has only been found on suspension-adapted cells, was underlined by a sub-cortical sheet of actin, forming a ball-like structure directly under the cell membrane; this actin conformation required re-organisation of the actin cytoskeleton from a typical fibrillar morphology in adherent cells. The actin content was higher in suspension cell lines compared to adherent cells, but actin up-regulation was also found in non-suspension adapted cells after they had been transferred into suspension. Sphere-like integrin beta 1 clustering on CCL61 grown in suspension could be induced by treatment with cytochalasin D, followed by suspension culture without the drug, however, despite the change in integrin beta 1 conformation these cells could not grow in suspension. The data suggests that adaptation to suspension growth requires conservation of integrins, presumably with respect to their role as structural elements anchoring the plasma membrane to the sub-cortical actin sheath, but it also requires additional changes in the interplay between integrin beta 1 and actin, for example, changes in the regulation of the associated proteins for successful suspension adaptation of CHO cells.
Supervisor: James, David C. Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.577424  DOI: Not available
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