Engineering Organized Epithelium Using Nanogrooved Topography in a Gelatin Hydrogel
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Engineering organized epithelium using nanogrooved topography in a gelatin hydrogel by John Paul Soleas, BMSc. A thesis submitted in conformity with the requirements for the degree of Master of Science Institute of Medical Science University of Toronto © Copyright by John P. Soleas, 2012 Engineering organized epithelium using nanogrooved topography in a gelatin hydrogel John Paul Soleas, BMSc. Master of Science, 2012 Institute of Medical Sciences University of Toronto Abstract Tracheal epithelium is organized along two axes: apicobasal, seen through apical ciliogenesis, and planar seen through organized ciliary beating, which moves mucus out of the airway. Diseased patients with affected ciliary motility have serious chronic respiratory infections. The standard method to construct epithelium is through air liquid interface culture which creates apicobasal polarization, not planar organization. Nanogrooved surface topography created in diffusible substrates for use in air liquid interface culture will induce planar organization of the cytoskeleton. We have created a nanogrooved gelatin device which allows basal nutrient diffusion. Multiple epithelial cells have been found to align in the direction of the nanogrooves in both sparse and confluent conditions. This device is also congruent with ALI culture as seen through formation of tight junctions and ciliogenesis. Thus, we have created nanogrooved surface topography in a diffusible substrate that induces planar alignment of epithelial cells and cytoskeleton. ii Acknowledgements I would like to thank my mentors, Drs. Alison P. McGuigan and Thomas K. Waddell for their patience, guidance, support, and for their example to always strive to be a better scientist. My future career aspirations as a physician and a scientist are due in no small part to this collaboration and their mentorship. I will forever be grateful to have had the opportunity to train under them. I am grateful for the friendship and collegial atmosphere of the McGuigan laboratory, the Waddell laboratory group, as well as the entire Latner Thoracic Surgery Research Laboratories. I would like to extend a special thanks to Dr. Siba Haykal for her valuable input, mentorship, and gifts of primary tracheal epithelium, and to Ms. Lily Guo for her valued input, and gift of primary tracheal epithelium. I thank Dr. Nadeem Moghal’s laboratory for their gift of and discussions on human tracheal epithelial cells. I thank my program advisory committee members, Drs. Craig Simmons, and Mingyao Liu for their insights and perspectives and critical review of this thesis. I thank the Canadian Institutes of Health Research and the Natural Science and Engineering Research Council of Canada for the Collaborative Health Research Project grant that made this collaboration possible and to the CIHR Training Program in Regenerative Medicine for their support through a graduate fellowship. Finally, I thank my family and friends for their unwavering support, love, and humour. iii Table of Contents Abstract ii Acknowledgements iii Table of Contents iv List of Figures and Tables xi List of Supplementary Figures xii List of Appendices xiii List of Abbreviations xiv Chapter 1 - Literature Review 1.1 Epithelial biology 2 1.1.1 Anatomy 2 1.1.2 Microstructure 3 1.1.3 Broad epithelial functions 4 1.1.4 Airway and respiratory epithelial function 5 1.1.4.1 Mucociliary clearance 8 1.2 Respiratory developmental biology 10 1.3 General epithelial polarization 13 1.4 Apical junctions 16 1.4.1 Tight junctions 16 1.4.1.1 Claudins 17 1.4.1.2 Occludins 18 1.4.2 Adherens junctions 19 iv 1.4.2.1 Cadherins 19 1.5 Hemidesmosomes 20 1.6 Cilia 22 1.6.1 Cilium structure 22 1.6.1.1 Transition zone 24 1.6.2 Ciliogenesis 25 1.6.2.1 FoxJ1 - Master program initiator 25 1.6.2.2 Timing 25 1.6.2.3 Basal body docking and nucleation 26 1.6.2.4 Intraflagellar transport 27 1.6.3 Primary cilium 27 1.6.4 Motile cilium 28 1.6.4.1 Mechanism of motion 28 1.6.4.2 Organization of cilia 29 1.7 Mechanobiology 30 1.8 Summative statement 34 Chapter 2 – Rationale, hypothesis, and aims 2.1 Rationale 36 2.1.1 Primary ciliary dyskinesia 37 2.1.2 How do we organize epithelium? From Soleas et al, 2012 38 2.1.2.1 Chemical signals 40 2.1.2.2 Mechanical signals 45 2.1.3 Study rationale 48 v 2.2 Hypothesis 49 2.3 Aims 49 Chapter 3 – Device Manufacture 3.1 Introduction 52 3.1.1 Replica moulding rationale 52 3.2 Process rationale 53 3.2.1 Holographic diffraction grating film 53 3.2.2 PDMS 54 3.2.3 Hydrogel choice 54 3.3 Methods 56 3.3.1 Generation of nangrooved PDMS mould 56 3.3.2 Collagen gel creation 56 3.3.3 Gelatin gel creation 57 3.3.4 Creating nanogrooved gel inserts 57 3.3.5 Scanning electron microscopy 58 3.3.6 Effect of scanning electron microscopy preparation on gelatin 59 hydrogel 3.3.7 Statistics 59 3.4 Results 61 3.4.1 Gelatin crosslinking optimization 61 3.4.2 Scanning electron microscopy 62 3.5 Discussion 65 3.5.1 Replication of PDMS 65 vi 3.5.2 Casting gelatin and moulding 65 3.5.3 Cell alignment on the nanogroove gel insert 66 3.5.4 Scanning electron microscopy 66 3.6 Conclusion 67 Chapter 4 – Respiratory epithelium on nanogrooved topography 4.1 Introduction 69 4.2 Rationale 69 4.3 Methods 70 4.3.1 Cell culture 70 4.3.2 Nanogroove PDMS seeding 70 4.3.3 Nanogroove gelatin seeding 71 4.3.4 Air liquid interface culture of BEAS-2B 71 4.3.5 Phase microscopy 71 4.3.6 Fluorescence microscopy 71 4.3.7 Quantification of cellular alignment 72 4.4 Results 73 4.4.1 Cell alignment on nanogrooves 73 4.4.1.1 ARPE19 73 4.4.1.2 IMCD3 77 4.4.1.3 BEAS-2B align on nanogrooves 81 4.4.1.4 Gelatin insert appears congruent with ALI 82 culture 4.5 Discussion 83 4.5.1 Epithelial cell alignment on nanogroove topography 83 vii 4.5.2 Response to topography 86 4.5.3 Substrate stiffness 88 4.5.4 Changing chemistry 90 4.5.5 BEAS-2B polarization on gelatin inserts 90 4.6 Conclusion 91 Chapter 5 –Future directions and conclusions 5.1 Introduction 93 5.2 Rationale 93 5.3 Methods 94 5.3.1 Human tracheal epithelia 94 5.3.2 Nanogroove culture 95 5.3.3 Air liquid interface culture 95 5.3.4 Immunohistochemistry 96 5.3.5 Imaging 97 5.3.6 Quantification of cellular alignment 97 5.37 Quantification of ciliated cells 98 5.4 Results 98 5.4.1 Human tracheal epithelial cells apicobasally polarize in 98 standard ALI culture 5.4.2 Human tracheal epithelial cells apicobasally polarize on gelatin 100 insert ALI culture 5.4.2 Alignment of primary human tracheal epithelial cell on nanogroove topography 102 viii 5.5 Future directions 103 5.6 Conclusion 105 Chapter 6 - Supplementary Figures Supplementary Macroscopic image of gelatin gels made from various 107 Figure 1 percentages of gelatin gel Supplementary Macroscopic image of 5% gelatin gels crosslinked 108 Figure 2 with various concentrations of glutaraldehyde Supplementary Bronchial epithelial cell line BEAS-2B on collagen 109 Figure 3 Supplementary Scanning electron micrographs of flat substrates 110 Figure 4 Supplementary National Institute of Health 3T3 fibroblasts grown on 111 Figure 5 nanogrooved substrates Supplementary Murine Inner Medullary Collecting Duct 3 (IMCD3) 112 Figure 6 epithelium on flat PDMS imaged using phase microscopy Supplementary Murine Inner Medullary Collecting Duct 3 (IMCD3) 113 Figure 7 epithelium on flat PDMS imaged using fluorescent microscopy Supplementary Human bronchial epithelial cell line (BEAS-2B) 114 Figure 8 sparsely seeded on nanogrooved substrates and imaged using phase microscopy Supplementary Human bronchial epithelial cell line (BEAS-2B) 115 Figure 9 sparsely seeded on nanogrooved substrates and ix imaged using fluorescent microscopy Supplementary Mean number of ciliated cells in control and gelatin 116 Figure 10 ALI inserts Chapter 7 – Appendix Appendix 1 Standard Operating Procedure for isolation of normal 118 human tracheal epithelial cells Chapter 8 – References 121 x List of Figures and tables Figure 1 Epithelial cell types found within the trachea, bronchioles and alveolus 6 Figure 2 Early morphogenesis of the foregut endoderm 10 Figure 3 The four major epithelial polarity domains are demarcated by various protein 14 complexes Figure 4 Ciliary structure 23 Figure 5 Examples of the tools of tissue engineering. 40 Figure 6 Specialized exemplar tools of epithelial tissue engineering 42 Figure 7 Generating nanogrooved hydrogels using a PDMS stamp 60 Figure 8 Scanning electron micrographs of nanogrooved substrates 63 Table 1 Contraction of gelatin hydrogel on the macro- and microscale 64 Figure 9 Sparse ARPE19 align on nanogroove topography 74 Figure 10 Confluent ARPE19 align on nanogroove topography 76 Figure 11 IMCD3 do not align morphologically on nanogroove topography 78 Figure 12 Confluent IMCD3 F-actin cytoskeleton aligns on nanogroove topography 80 Figure 13 Confluent normal human bronchial epithelial cell line BEAS-2B grown on 81 nanogrooves have aligned F-actin cytoskeleton Figure 14 Primary cilia are present on BEAS-2B differentiated on gelatin gels 83 Figure 15 Human tracheal epithelial cells form tight junctions and multiciliated cells 99 during standard ALI culture Figure 16 Human tracheal epithelial cells form tight junctions and multiciliated cells 101 during ALI culture on a gelatin filter Figure 17 Human tracheal epithelial cells seeded on nanogrooves do not align 101 xi List of Supplementary Figures Supplementary 1 Macroscopic image of gelatin gels made from various 107 percentages of gelatin gel Supplementary 2 Macroscopic image of 5% gelatin gels crosslinked with various 108 concentrations of glutaraldehyde.