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Design of Bioreactors for Mesenchymal Stem Cell Tissue Engineering

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Design of Bioreactors for Mesenchymal Stem Cell Tissue Engineering Design of Bioreactors for Mesenchymal Stem Cell Tissue Engineering Pankaj Godara A Thesis Submitted for the Degree of Doctor of Philosophy at the University of New South Wales Faculty of Engineering Graduate School of Biomedical Engineering 2010 Originality Statement ‘I hereby declare that this submission is my own work and to the best of my knowledge it contains no materials previously published or written by another person, or substan- tial proportions of material which have been accepted for the award of any other degree or diploma at UNSW or any other educational institution, except where due acknowl- edgement is made in the thesis. Any contribution made to the research by others, with whom I have worked at UNSW or elsewhere, is explicitly acknowledged in the thesis. I also declare that the intellectual content of this thesis is the product of my own work, except to the extent that assistance from others in the project’s design and conception or in style, presentation and linguistic expression is acknowledged.’ Signed ...................................................... Date ...................................................... Copyright Statement ‘I hereby grant to the University of New South Wales or its agents the right to archive and to make available my thesis or dissertation in whole or part in the University li- braries in all forms of media, now or hereafter known, subject to the provisions of the Copyright Act 1968. I retain all proprietary rights, such as patent rights. I also retain the right to use in future works (such as articles or books) all or part of this thesis or dissertation. I also authorise University Microfilms to use the abstract of my thesis in Dissertations Abstract International (this is applicable to doctoral theses only). I have either used no substantial portions of copyright material in my thesis or I have obtained permission to use copyright material; where permission has not been granted I have applied/will apply for a partial restriction of the digital copy of my thesis or dissertation.’ Signed ...................................................... Date ...................................................... Authenticity Statement ‘I certify that the Library deposit digital copy is a direct equivalent of the final offi- cially approved version of my thesis. No emendation of content has occurred and if there are any minor variations in formatting, they are the result of the conversion to digital format.’ Signed ...................................................... Date ...................................................... Acknowledgements First and foremost I would like to thank my family, my mother Saroj, my father Lal, and my brother Vikas for their constant encouragement, endless love, and unwavering support. Thank you for always believing in me and for making me the person I am today. I survived and the sky did not fall. This body of work would not have been possible without kind assistance from my supervisors. Thank you to Bruce Milthorpe and the Graduate School of Biomedical Engineering for giving me the opportunity to undertake this project, and to Clive McFarland for contributions. I would like to express my most sincere heartfelt gratitude to Robert Nordon for his patient supervision and guidance in seeing this project through to the end. Thank you for your help in the preparation of this manuscript, for taking such an inspiring interest in my work, and for teaching me so much. What an experience. John Whitelock is thanked for advice and friendship, especially during the latter stages of this project. Research students Joost vab den Berg and Carl Gabel are acknowledged for assistance with experimental work, and Andrew Sims is thanked for help with all things Latex, Matlab and CAD. NIH 3T3 cells were kindly provided by iNano, University of Aarhus, Aarhus, Denmark. MS-5 cells were kindly donated by Alla Dolnikov of the Children’s Cancer Institute Australia, and equine bone marrow was kindly donated by Chris O’Sullivan of the Randwick Equine Centre. To all the GSBmE L4 technical staff, thank you for your advice and guidance. To all the past and present research students at GSBmE, thank you for enriching this journey. To my dear friends Gina Katsiolis, Richa Gupta and Thania Kearns - Thank you for always lending an ear to listen; for your patience and kindness; for being the voice of reason when there was none; and for reminding me of life beyond this thesis. A clay pot sitting in the sun will always be a clay pot. It has to go through the white heat of the furnace to become porcelain. Mildred W. Struven Publications and presentations arising from this thesis Journal Papers 1. Godara, P., Nordon, R.E., McFarland, C.D. Mesenchymal stem cells in tissue en- gineering. Journal of Chemical Technology & Biotechnology, Volume 83, Number 4, April 2008 , pp. 397-407(11 pages) 2. Godara, P., McFarland, C.D., Nordon, R.E. Design of bioreactors for mesenchy- mal stem cell tissue engineering. Journal of Chemical Technology & Biotechnol- ogy, Volume 83, Number 4, April 2008 , pp. 408-420(13 pages) Conference Papers 1. Nordon, R. E., The, O., Godara, P., You, S., Van Den Berg, J. and Rosengarten, G. Membrane Culture System for Manufacture of Cells for Transplantation: From Lab to Clinic. 19th Annual Conference of Australasian Society for Biomaterials and Tissue Engineering, 21-23 January 2009, Sydney, Australia. 2. Godara, P., Ko, K., You, S., Soon, L., Foong, F., Rosengarten, G., Nordon, R. In Vitro Methods for Investigation of Cell-Matrix Interactions. Matrix Biology So- ciety of Australia and New Zealand Annual Meeting, 13-16 October 2008, Mantra Ettalong Beach, Australia. 3. Godara, P., Nordon, R.E., Ko, KH., Milthorpe, B.K. and McFarland, C.D. Iden- tification of Adult Stem Cells for Tissue Engineering Applications. ISAC Samuel A. Latt Meeting: Stem Cells in the Age of Fluorescence Technology, Australian Stem Cell Centre Conference, 6-9 November 2005, Gold Coast, Australia. 4. Godara, P., Nordon, R.E., Ko, KH., Milthorpe, B.K. and McFarland, C.D. Iden- tification of Adult Stem Cells for Tissue Engineering Applications. Stem Cell and Tissue Engineering Common Interest Group Mini Symposium, August 18 2005, Sydney Australia. 5. Godara, P., Nordon, R.E., Ko, KH., Milthorpe, B.K. and McFarland, C.D. Iden- tification of Adult Stem Cells for Tissue Engineering Applications. Sydney Tissue Engineering and Matrix Group IXth Symposium, May 12 2005, Sydney, Australia. 6. Godara, P., Nordon, R.E., Ko, KH., Milthorpe, B.K. and McFarland, C.D. Iden- tification of Adult Stem Cells for Tissue Engineering Applications. Australian Society for Biomaterials Conference, March 31-April 2 2005, Victor Harbour, Australia. Abstract The ex vivo manufacture of functional organs and tissues for implantation can impact on therapeutic needs arising from an ageing population. The rapid expansion of the field of tissue engineering has arisen in response to this clinical need. In order for mesenchymal stem cells (MSC) to be useful clinically, sufficient numbers must be obtained. It was hypothesised that MSC could be isolated from a heterogeneous population of cells, and that hollow fibre bioreactor systems may be a scalable technology for expansion. In order to manufacture functional tissues, appropriate bioreactor devices must be developed to direct cellular differentiation. It was hypothesised that long-term ex vivo tissue gradients could be established. A substrate (Aldefluor) for aldehyde dehydrogenase (ALDH) activity was employed to label cells from human umbilical cord blood (hUCB) and cells isolated from rat, murine, porcine and equine origins. Growth of anchorage dependent cells was carried out in cuprophan hollow fibres coated with a novel recombinant protein, with and without extracapillary co-culture. Micro bioreactors were manufactured using the techniques of lithography from polydimethylsiloxane, and device biocompatibility was assessed using the anchorage dependent cell line NIH 3T3. While cells isolated from rat, murine, porcine and equine origins showed an ALDH bright population, MSC could not be purified from hUCB. Cellular attachment to cel- lulose based substrates coated with the recombinant protein occurred within 2 hours. Anchorage dependent cells could not be maintained in the hollow fibres. Stable con- centration gradient profiles were generated experimentally in two micro-devices with differing geometries. In the second device, the concentration gradient was maximal in the region of flow stasis, and cells were found to remain viable inside the cell chamber belowaflowrateof4μL/min for 72 hours. Aldefluor substrate was not useful in the prospective isolation of MSC from a heteroge- neous population. Coating of cellulose substrates with a novel recombinant protein was found to be necessary for cell attachment. Growth in the hollow fibres was found to be suboptimal compared to conventional methods. Cell chemotaxis and tissue morphogen- esis could be studied using the second micro-device developed without the confounding effect of fluid shear stress. Table of Contents Abbreviations and Symbols x List of Tables xiii List of Figures xiv 1 Introduction 1 1.1 Research Motives ............................... 1 1.2ThesisAims.................................. 3 1.3ThesisLayout................................. 4 2 Literature Review 5 2.1Introduction.................................. 5 2.2TissueEngineering.............................. 5 2.3StemCells................................... 8 2.3.1 Definitionsandterminology....................
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