Adaptation to suspension growth: analysing the surface of suspension growth adapted Chinese hamster ovary cells by Christa G Walther submitted for the degree of Doctor of Philosophy Department of Chemical and Biological Engineering January 2013 Analysing the surface of suspension growth adapted CHO cells Statement of Originality: The work presented in this thesis is to the best of my knowledge and belief original, except as acknowledged in the text. I hereby declare that I have not submitted this material, either in whole or in part, for a degree at this or any other institution. January 2013 -2- Analysing the surface of suspension growth adapted CHO cells dedicated to my mum Rita Wolters * 1932 † 2009 Du fehlst. -3- Analysing the surface of suspension growth adapted CHO cells Acknowledgement I would like to thank Prof. David C. James and Dr. Mark Leonard for the opportunity to work on this project and learn about bio-pharmaceutical production in CHO cells. I am grateful for the feedback and input I got from Dr. Robin Heller-Harrison and Dr. Mark Melville, both previously at Wyeth (now Pfizer), during the first two years of the project. Also I would like to thank Pfizer for the financial support. Rob Whitfield, who has been using the same cell lines in his PhD project, was always helpful with tips on cell culture, cell splitting during times of holiday and organised the continuous flow of the proprietary media by sending lots of emails and handling the huge delivery boxes. For the supply with the media a big thank you goes as well to the media production department at Pfizer and to Steve Brennan for organising the shipments. I am really grateful for the help I got at the Sheffield Medical School from Sue Newton and Kay Hopkinson, who answered every question about flow cytometry I had and also found alternative instruments to measure on when need arose. At the Kroto Research Institute, Dr. Nicola Green introduced me to confocal microscopy, trained me patiently on the use of the confocal microscope and helped with all questions I had. I enjoyed working with the different nationalities and personalities in the group; to Olivia Mozley a big thank you for understanding my English when I myself wasn't too sure what I was trying to say and for discussing confocal experiment set-up. More thanks go to Dr. Paul Dobson, who undertook the effort to read a thesis draft, and to Dr. Henriette Jensen, Dr. Esther Karunakaran and Dr. Phil Jackson for their feedback on single chapters. I would also like to thank my sister, my father and my friends in Germany who, although they found the project rather unexciting, continued to be interested in my progress. Finally, I would like to thank my husband Thomas, who besides doing a lot of cooking over the last years to keep us alive (he is now a much better cook than I am), also tried hard to learn and understand major biological terms in order to perhaps become able to understand my project. He also made sure that typical German grammar mistakes have been kept to a minimum. I promise him to not be as short tempered next year as during the last months. -4- Analysing the surface of suspension growth adapted CHO cells 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. -5- Analysing the surface of suspension growth adapted CHO cells Table of Contents List of Tables ............................................................................................................................... 11 List of Figures ............................................................................................................................. 12 Abbreviations and Synonyms........................................................................................................15 1. Chapter Introduction ................................................................................................................................. 17 1.1 Basic background .............................................................................................................. 17 1.2 Objectives and Approaches used ....................................................................................... 19 1.3 Summary of the main results ............................................................................................ 20 2. Chapter Chinese hamster ovary cells as a production vehicle that requires suspension adaptation ........ 22 2.1 Chinese hamster ovary cells for bio-pharmaceutical production ...................................... 22 2.2 Cell surface changes as a reaction to a new environment ................................................. 24 3. Chapter Experimental techniques ............................................................................................................ 31 3.1 Cell lines ........................................................................................................................... 31 3.2 Cell culture conditions ...................................................................................................... 31 3.2.1 Suspension culture ..................................................................................................... 31 3.2.2 Adherent culture ........................................................................................................ 21 3.3 Cell preparation for flow cytometry ................................................................................. 32 3.4 Cell preparation for confocal microscopy ......................................................................... 32 3.5 Flow cytometry – instrumentation set-up ......................................................................... 33 3.6 Confocal microsopy - instrumentation set-up ................................................................... 33 3.7 Actin labelling ................................................................................................................... 33 3.7.1 Actin: flow cytometry staining .................................................................................. 33 3.7.2 Actin: confocal microscopy staining ......................................................................... 34 3.8 Integrin beta 1 labelling .................................................................................................... 34 3.8.1 Integrin beta 1: flow cytometry staining ................................................................... 34 3.8.2 Integrin beta 1: confocal microscopy staining .......................................................... 34 3.9 Integrin alpha 1 labelling .................................................................................................. 35 3.9.1 Integrin alpha 1: flow cytometry staining ................................................................. 35 -6- Analysing the surface of suspension growth adapted CHO cells 3.9.2 Integrin alpha 1: confocal microscopy staining .......................................................