Donaldson, Amy Rose

271 Pages·2017·6.26 MB·English

by  Christine Middleton

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
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