Development of Biomimetic Platforms to Investigate the Influence of the Extra-Cellular Environment on Immunological Responses Amy Rose Donaldson Thesis submitted to the University of Nottingham for the degree of Doctor of Philosophy Immunology Department, School of Life Sciences, University of Nottingham August 2016 Abstract The immune system comprises highly sophisticated networks of cells and signalling molecules which function in concert to protect the body against pathogens. Within this system a role for the extra-cellular microenvironment as a crucial mediator of immune responses is becoming increasingly apparent. Conventional in vitro cultures lack physiologically relevant extra-cellular cues, such as extracellular matrix (ECM) and shear flow. Tissue engineering can be used to simulate features of the natural microenvironment for the development of biologically relevant platforms. It is anticipated that this will enable the study of the influence of the extra-cellular environment on immune responses. This thesis describes the development and characterisation of tissue-engineered platforms for immune cell culture which incorporate the ECM and shear flow. This work goes on to apply these platforms for the study of the effect of the extra-cellular environment on dendritic cells and their interactions with T cells in the context of immunological stimulation. The ECM defines the three-dimensional architecture of the natural microenvironment. It provides structural support and also promotes cell motility in tissues. This is important for the function of the immune system as it directs the organisation and interactions of immune cells which ultimately contributes to the modulation of immune responses. I Candidate synthetic and natural biomaterials were assessed for their suitability to provide an in vitro extracellular matrix (ECM) platform for human immune cell culture. The suitability of these materials to provide an artificial ECM platform was based on the viability, resting immune state and immune competence of the cells. The synthetic biomaterials tested were a thermo-responsive colloidal gel and electrospun PET and PLGA scaffolds coated with a thermo-responsive polymer. An important finding from the work done with the colloidal gel was that the human dendritic cells, which were incorporated into the gel at the beginning of the experiment, could not be separated from the material for flow cytometric analysis. Therefore, characterisation of the colloidal gel for immune cell culture could not be completed. Regarding the characterisation of the electrospun PET and PLGA scaffolds, although they did not significantly impair cell viability of dendritic cells they were found to induce cell maturation. As a result, none of the synthetic biomaterials were found to be a suitable ECM surrogate. A semi-natural biomaterial, gelatin methacryloyl (GelMA) hydrogel, was included in the investigation. The results from the characterisation of GelMA for human immune cell culture indicated that the hydrogel induced a pro- inflammatory immune response due to the profile of secreted cytokines. Based on this, GelMA was also discounted as an appropriate material for the development of the ECM platform. II The final ECM candidate was a collagen hydrogel, which is a naturally-derived biomaterial. The collagen hydrogel was shown to support immune cell survival and human dendritic cells maintained an immature phenotype in culture. In addition, typical responses to immunological stimuli by human dendritic cells and T cells were observed in collagen hydrogel cultures. This work demonstrated that out of the biomaterials which were characterised, the collagen hydrogel was the most suitable biomaterial for the development of the ECM platform. The influence of the collagen hydrogel ECM platform on antigen-specific immune responses was investigated in the context of autologous human dendritic cell and T cell co-cultures stimulated with the model antigen Mycobacterium tuberculosis purified protein derivative, also referred to as PPD. The results from these experiments indicated that the presence of the collagen hydrogel increased the sensitivity and specificity of the immune response, compared to conventional tissue culture conditions. An attempt was made at utilising the ECM platform to investigate immune responses to chemical sensitisers to address the requirement for in vitro alternatives to replace current animal testing methods. In this work, innate and adaptive immune responses to sensitisers were detected using the ECM platform. However, the reproducibility of these experiments was low due to large donor variation. Therefore the effect of the ECM platform on immune responses to sensitisers could not be evaluated. This difficulty likely reflects the complexity of the molecular and cellular mechanisms which lead to the acquisition of chemical sensitisation. III Shear flow is a type of physiological stress to which immune cells are exposed in vivo due to the movement of blood and lymph fluid. Recent studies have implicated flow as an immunologically relevant stimulus, capable of inducing changes in the expression of receptors and chemokines involved in regulating immune cell migration, and activating immune receptor signalling. A fluidic cell culture platform was developed to recapitulate the effect of shear flow. Two different prototypes were constructed, one of which was taken forward and characterised for immune cell culture applications. The fluidic platform taken forward had a paper-based cell culture scaffold which was coated with collagen hydrogel. The scaffold was found to induce maturation of human dendritic cells which was attributed to the possibility of incomplete coverage of the scaffold by the collagen hydrogel. The viability of dendritic cells was slightly impaired by flow, however not significantly. Interestingly, when exposed to shear flow, dendritic cells maintained a less mature phenotype compared to their static counterparts. Antigen-specific immune responses were studied on the fluidic platform by setting up co-cultures comprising PPD-stimulated autologous human dendritic cells and T cells. Typical T cell activation was observed on the platform and the sensitivity and specificity of immune responses was found to be greater under flow conditions, compared with static cultures. IV In conclusion, this thesis demonstrates the value of developing biomimetic platforms for studying the influence of the extra-cellular environment on immune responses. Finally, the ability to mimic extra-cellular cues to which cells are exposed in vivo has the potential to generate more realistic immune responses in the lab. This presents huge opportunities for advancing understanding in immunology. It also has implications for methods used in research, drug discovery and safety testing, where currently only animals provide a representative system for the study of immune reactions. It is anticipated that enhancing the physiological relevance of in vitro cell culture will ultimately contribute to the reduction of animals used in research and testing. V Acknowledgements To begin, I would like to thank my academic supervisors, particularly Professor Amir Ghaemmaghami for giving me the opportunity to work under his supervision and for his support throughout my PhD. Many thanks also to my co-supervisors Professor Cameron Alexander and Doctor Felicity Rose for their expert opinions on and guidance of my research. I would like to acknowledge those who have assisted me with my experimental work. Thanks to Doctor David Onion and Nikki Croxall for their support in flow cytometry; Laurence Hall for his contribution to the gene expression studies; Christie Grainger-Boultby for her help with SEM; Doctor Tim Self for training and advice on the confocal microscope, and to all of the members of the Polymer Therapeutics Group at the University of Nottingham, particularly Afnan Aladdad and Shwana Braim for providing some of the materials for my work. I wish to acknowledge the external collaborators from the Ziaie Biomedical Microdevices Lab, Perdue, USA, particularly Rahim Rahimi for the fluidic devices and technical guidance he provided. I am grateful for the opportunities I have had had during my PhD. Particularly the valuable experience I gained from my visits to the Khademhosseini Lab, MIT, Boston, USA. I would like to thank Doctors Mehdhi Nikkhah and Waseem Asghar for their support during the time I spent there. VI I would like to acknowledge the NC3Rs for providing the financial support which made this project possible. I wish to thank all of the past and present members of the Allergy and Tissue Modelling Group. In particular Doctor Fabian Salazar, Doctor Sonali Singh, Doctor Wejdan Aldajani, Su Su Htwe, Raudzah Kamal, Asha Hassan, Shehnaz Ahmed, Hassan Rostam and Doctor Paul Cato. You’ve all been a constant source of support and inspiration. Thank you to my friends Ellie, Nicola, Laura and Lucinda for your support and for persuading me to occasionally take a break from work! To all of my family, the biggest thank you, especially to my grandparents who have done so much for me and to my sister and best friend Kate, who has been there for me through all of the highs and lows. Last, but not least, to my parents, for your unconditional love and support, thank you. Without your belief in me I would never have achieved what I have. I wish to dedicate this thesis to my uncle Andy, I miss you every day and hope you would have been proud. Lots of love. VII Contents ABSTRACT ................................................................................................................................ I ACKNOWLEDGEMENTS.................................................................................................... VI CONTENTS ......................................................................................................................... VIII TABLE OF FIGURES ............................................................................................................ XI TABLE OF TABLES ............................................................................................................. XV ABBREVIATIONS .............................................................................................................. XVI CHAPTER 1 INTRODUCTION ............................................................................................. 1 1.1. CONSIDERING HOW THE MICROENVIRONMENT INFLUENCES IMMUNITY ..................... 1 1.2. INTRODUCING DENDRITIC CELLS ............................................................................... 2 The Role of Dendritic Cells in Immunity ............................................................................ 5 Dendritic Cells in Contact Allergy ..................................................................................... 9 1.3. LYMPH NODES ARE SPECIALISED TO CHOREOGRAPH INTERACTIONS BETWEEN DENDRITIC CELLS AND T CELLS ........................................................................................... 15 1.4. EXTRA-CELLULAR INFLUENCES ON DENDRITIC CELLS ............................................ 18 1.5. APPLYING TISSUE ENGINEERING TO SAFETY TESTING ............................................. 21 Methods for Predicting Chemical Sensitisation ............................................................... 23 1.6. THESIS PLAN ............................................................................................................ 37 CHAPTER 2 CHARACTERISATION OF THE IMMUNE-COMPATIBILITY OF BIOMATERIALS ................................................................................................................... 39 2.1. INTRODUCTION ......................................................................................................... 39 Chapter Overview ............................................................................................................ 39 Immune Responses to Biomaterials .................................................................................. 41 Biomaterials for Tissue Engineering ................................................................................ 43 Chapter aims .................................................................................................................... 52 VIII 2.2. MATERIALS AND METHODS ..................................................................................... 53 2.3. RESULTS AND DISCUSSION ....................................................................................... 70 Investigating Synthetic Biomaterials for Immune Cell Culture ........................................ 70 Naturally-derived Biomaterials ........................................................................................ 81 2.4. CONCLUSIONS AND FURTHER WORK ....................................................................... 98 CHAPTER 3 DESIGN, FABRICATION & CHARACTERISATION OF A FLUIDIC PLATFORM FOR IMMUNE CELL CULTURE .............................................................. 107 3.1. INTRODUCTION ............................................................................................................. 107 Chapter Overview .......................................................................................................... 107 Immune Modulation by Fluid Shear Stress .................................................................... 108 Considerations for the Design & Development of Fluidic Platforms ............................ 111 Chapter Aims .................................................................................................................. 115 3.2. MATERIALS AND METHODS .......................................................................................... 116 3.3. RESULTS AND DISCUSSION ........................................................................................... 122 Construction of Fluidic Platforms for Cell Culture ....................................................... 122 Characterisation of a Paper-Based Scaffold for Immune Cell Culture .......................... 127 Characterisation of Fluidic Platform with a Paper Scaffold for Immune Cell Culture . 134 3.4. CONCLUSIONS AND FURTHER WORK ............................................................................ 144 CHAPTER 4 APPLICATION OF PHYSIOLOGICALLY RELEVANT PLATFORMS FOR THE STUDY OF ANTIGEN-SPECIFIC DENDRITIC CELL – T CELL INTERACTIONS .................................................................................................................. 149 4.1. INTRODUCTION ............................................................................................................. 149 Chapter Overview .......................................................................................................... 149 Cell-Mediated Adaptive Immunity .................................................................................. 151 T Cell Activation ............................................................................................................ 152 Chapter Aims .................................................................................................................. 160 4.2. MATERIALS AND METHODS .......................................................................................... 161 4.3. RESULTS AND DISCUSSION ........................................................................................... 163 Optimising Antigen-Specific T cell Activation In Vitro .................................................. 163 IX