DEVELOPMENT OF A NOVEL 3D NANOFIBRE CO-CULTURE MODEL FOR CHARACTERISATION OF NEURAL CELL DEGENERATION Joseph Mathew Chemmarappally (N0404816) A thesis submitted in partial fulfilment of the requirements of Nottingham Trent University for the degree of Doctor of Philosophy Supervisor team: Prof Bob Stevens, Dr Luigi De Girolamo & Dr Alan Hargreaves October 2019 DECLARATION OF OWNERSHIP This submission is the result of my work. All help and advice, other than that received from tutors, has been acknowledged and primary and secondary sources of information have been properly attributed. Should this statement prove to be untrue, I recognise the right and duty of the Board of Examiners to recommend what action should be taken in line with the University’s regulations on assessment contained in the Handbook Sign.................................... Date.....28/10/2019.................... Copyright Statement This work is the intellectual property of the author, and may also be owned by the research sponsor(s) and/or Nottingham Trent University. You may copy up to 5% of this work for private study, or personal, non-commercial research. Any re-use of the information contained within this document should be fully referenced, quoting the author, title, university, degree level and pagination. Queries or requests for any other use, or if a more substantial copy is required, should be directed in the owner(s) of the Intellectual Property Rights. Dedication I want to dedicate my research to my loving grandfather Mr. Thomas Chandy Konnath, who sadly passed away due to Parkinson’s disease. He is the reason I choose to pursue research on neurodegenerative disease, and I hope to make a difference, even in the smallest way possible. Acknowledgements First, I want to sincerely thank my director of studies Prof Bob Stevens, for constant support and guidance throughout my PhD journey. I cannot thank enough Dr Luigi De Girolamo (Gino) and Dr Alan Hargreaves for giving me this fantastic opportunity to work alongside them. I am immensely grateful to Bob Stevens for being enthusiastic, motivating, providing thoughtful advice, being an inspiration and for being my mentor. Thanks to Dr Gino for being very supportive through every possible moment for the past 9 years and for becoming a good friend. My sincere thanks to Dr Alan for constant expert advice, guidance and support in this challenging journey. I would also like to express my gratitude to NTU QR funding, Vice Chancellor bursary and the Physics department NTU for funding my PhD studentship. A special thanks to the Physics department and IBRC technical support team Daniel Yates, Christopher Mutton, Ryan Toms, Dr Jacob Spears, Dave Parker and Dr Kathryn Kroon for all the spontaneous help and support without any hesitation. A big thanks to Dr Sammy Cheung for helping me and training me on OCT imaging. Dr Neranga Abeywickrama, thank you for educating me on many types of equipment, helping me out every time I wanted something and for putting up with me for seven years. Dr Enzo Fornair, Dr Costas Tsakonas, Dr Demosthenes Koutsogeorgis and Dr Nikolaos Kalfagiannis thank you so much for the help and support at times and for all the fun times and laughs at iSMART. James, Jodie and Cristina, I have known you guys only for a short time but you guys have made an impact and thanks for the support at times. A very big thank you to my closest friend Henry Pegram and Michael Cripps for all the fantastic time, motivation and for supporting me like family during this journey, you both have made this journey so easy. This would be incomplete without all my dear fantastic friends/PhD colleagues at NTU for their company and support during hard times especially, Dan, Magda, Pauline, Sarah, Anna, Anahita, Simon, Devika, Nick, Awais, Jordan, Laura, Falguni, Elisa, Grace, Charlotte, Veeru and Brijesh. Also, a big thank you to my lovely friends back in India for the push and support every time I needed one. This PhD journey has not only given me knowledge and skill but also beautiful and wonderful memories that I would cherish forever. I must thank my lovely, sweetest best friend, my full-time accomplice and my inspiration, Dr Divya Nagarajan, for being with me through all the happiest and saddest moments. None of this would have happened if she was not there to guide me through the right path. She pushed me from being an average student to a PhD holder and made me what I am today. My path would have stayed lost without her. Finally, a very big thank you to my Lovely dad, my sweet mom, my caring sister and a supportive brother-in-law for helping me and supporting me through every possible situation to achieve my goal. I hope I have made you all proud. Table of Contents LIST OF FIGURE .......................................................................... I LIST OF TABLES ......................................................................... V ABBREVIATIONS ....................................................................... VI ABSTRACT ................................................................................. IX CHAPTER 1: MAIN INTRODUCTION ............................................ 1 1.1 Electrospinning ......................................................................................... 1 1.1.1 Nanofibres ............................................................................................ 2 1.1.2 Polyacrylonitrile ..................................................................................... 3 1.1.3 Polyetheramines .................................................................................... 5 1.1.4 Nanofibres for In vitro model .................................................................. 6 1.1.5 Application of nanofibres in bioscience ...................................................... 7 1.1.6 Nanofibre and tissue engineering ............................................................. 9 1.2 Neurodegenerative Disease .................................................................... 13 1.2.2 Midbrain hub of motor movements ......................................................... 16 1.2.2.1 Neurons ....................................................................................... 17 1.2.2.2 Glial Cells ..................................................................................... 18 1.2.2.3 Role of Astrocytes .......................................................................... 19 1.2.4 Neurotoxins ........................................................................................ 23 1.2.4.1 Rotenone ...................................................................................... 24 1.2.4.2 MPTP ........................................................................................... 24 1.2.4.3 MG132 ......................................................................................... 24 1.2.4.4 BSO ............................................................................................. 25 1.3 In vitro cell Models for neural cell degeneration ..................................... 25 1.3.1 Toxin Models....................................................................................... 26 1.3.1.1 6-hydroxydopamine (6-OHDA) ........................................................ 27 1.3.1.2 1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine (MPTP) ........................ 27 1.3.1.3 Rotenone ...................................................................................... 27 1.3.2 Genetic Models .................................................................................... 29 1.3.3 Pre-clinical models of neural cell degeneration ......................................... 29 1.4 3D Cell Culture ........................................................................................ 32 1.4.1 Cellular Spheroids ............................................................................... 34 1.4.2 Hydrogel models ................................................................................. 36 1.4.3 Engineering based scaffolds for neural tissue construction ......................... 37 1.4.4 Microfluidic technology models .............................................................. 38 1.5 Novelty of This Study .............................................................................. 40 1.5.1 Structure of this study ......................................................................... 41 CHAPTER 2: MATERIALS AND METHODS................................... 43 2.1 Materials ................................................................................................. 43 2.1.1 Cell Culture ........................................................................................ 43 2.1.1.1 Cell Lines ...................................................................................... 43 2.1.1.2 Cell Culture Reagents ..................................................................... 43 2.1.1.2.1 Cell Culture Media .................................................................... 43 2.1.1.2.2 Common Cell Culture Reagents .................................................. 44 2.1.2 Plastic, Glassware and other materials .................................................... 44 2.1.3 Chemicals and reagents ....................................................................... 45 2.1.4 Equipment .......................................................................................... 47 2.1.5 Antibody ............................................................................................ 50 2.1.5.1