Integrating Advanced 3D Cell Culture Techniques with Rapid
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INTEGRATING ADVANCED 3D CELL CULTURE TECHNIQUES WITH RAPID- PROTOTYPING MICROFLUIDICS FOR TRANSLATIONAL APPLICATIONS BY SHINY AMALA PRIYA RAJAN A Dissertation Submitted to the Graduate Faculty of WAKE FOREST UNIVERSITY GRADUATE SCHOOL OF ARTS AND SCIENCES In Partial Fulfillment of the Requirements For the Degree of DOCTOR OF PHILOSOPHY Biomedical Engineering December 2019 Winston-Salem, North Carolina Approved By: Dr. Adam R. Hall, Ph.D., Advisor Dr. Aleksander Skardal, Ph.D., Co-Advisor Dr. Konstantinos Votanopoulos, M.D., Ph.D., Chair Dr. Jennifer Munson, Ph.D. Dr. Scott Verbridge, Ph.D. “..with God all things are possible.” Matthew 19:26 To my mom and my sister, who has always supported my dreams, To Julio, who helped those dreams come true, and To everyone, who motivated this work. ii TABLE OF CONTENTS DEDICATION ii LIST OF FIGURES AND TABLES vi LIST OF ABBREVIATION ix ABSTRACT 1 1. Chapter 1- Introduction 3 1.1. Why 3D cell culture? 3 1.2. Types of 3D cell culture techniques 8 1.2.1. Scaffold-free culture technique 9 1.2.2. Scaffold-based culture technique 13 1.3. Interfacing 3D cell culture with microfluidic systems 18 1.3.1. Current microphysiological systems 22 1.3.2. Adhesive-film based microfluidics 24 1.4. Micro-engineered models 27 1.4.1. 3D Organ models 29 1.4.2. 3D Disease models 32 1.5. Conclusion 36 Reference 40 2. Chapter 2 – Development of an integrated multi-tissue adhesive film-based Organ-on-a-chip platform for assessing drug efficiency and toxicity 57 Abstract 60 2.1. Introduction 61 2.2. Experimental Methods 64 2.3. Results and Discussion 76 2.3.1. Implementation of a high viability 3-organoid platform with drug assessment 76 2.3.2. Implementation of a high viability 6-organoid platform with drug assessment 83 2.4. Conclusion 94 iii Reference 97 3. Chapter 3 – Developing an in vitro model of patient-derived 3D tumor Organoids and integrate with microfluidics to facilitate patient-centric therapeutic screening 106 Abstract 108 3.1. Introduction 110 3.2. Materials and Methods 114 3.3. Results 126 3.3.1. Biospecimen processing, organoid production, and assessment of patient-specific HNSCC tumor organoids 126 3.3.2. Comparison of cancer treatment responses between sponge assay and PDOs 132 3.3.3. Validation of β-Lapachone-mediated cytotoxicity on the PDOs 137 3.3.4. Biospecimen processing, organoid production, and assessment of patient-specific mesothelioma tumor organoids in an AFB microfluidic chip 142 3.3.5. In vitro chemotherapeutic drug screens correlate with subject drug response 145 3.4. Discussion and Conclusion 148 Reference 153 4. Chapter 4 – Multi-domain photo-patterned 3D tumor constructs in a micro- physiological system for analysis, quantification, and isolation of infiltrating Cells 161 Abstract 163 4.1. Introduction 164 4.2. Experimental Methods 166 4.3. Results and discussion 176 4.3.1. Interstitial flow 176 4.3.2. Effect of hydrogel matrix stiffness on tumor cell migration 179 4.3.3. Influence of the chemotherapeutics agents 5FU on HCT-116 Infiltration 182 iv 4.3.4. Influence of the chemotherapeutics agents Marimastat on HCT-116 infiltration 187 4.3.5. Selective hydrogel dissolution to isolate migrated cells 190 4.4. Conclusion 193 Reference 196 5. Chapter 5 – Development of a patient-specific 3D invasion model to improve therapy outcomes of cancer patients 203 Abstract 205 5.1. Introduction 206 5.2. Experimental methods 208 5.3. Results and discussion 213 5.3.1. Influence of chemotherapeutic agent on infiltrating cells in a patient-derived mesothelioma tumor organoid 213 5.3.2. Infiltrated GBM cells display markers of invasive and mesenchymal phenotype 215 5.4. Conclusion 218 Reference 220 6. Chapter 6 – Conclusion and future direction 224 6.1. Discussion and Conclusion 224 6.2. Future direction 231 Reference 235 Appendix 236 Curriculum Vitae 265 v LIST OF FIGURES AND TABLES Figure 1.1 Adhesive, topographical, mechanical, and soluble cues in 2D and 3D 5 Figure 1.2. Contact surface of different cell culture methods 6 Figure 1.3 Common 3D cell culture techniques 11 Figure 1.4. Microfluidic techniques for 3D cell culture 21 Figure 1.5. Fabrication of PDMS based microfluidic device 23 Figure 1.6. Adhesive film based Microfluidic chip 26 Figure 1.7. The influence of the tumor microenvironment in cancer progression 34 Figure 2.1. In situ photopatterning of multiple organ-specific tissues 65 Figure 2.2. Drug toxicity assessment of capecitabine in a 3-organoid system 80 Figure 2.3: Viability quantification under insult by capecitabine in a 3-organoid System 82 Figure 2.4. Drug toxicity assessment of cyclophosphamide in six-organ system 86 Figure 2.5. Viability quantification under insult by cyclophosphamide in a 6- organoid system 88 Figure 2. 6. Drug toxicity assessment of ifosfamide in a 6-organoid system 91 Figure 2.7. Viability quantification under insult by ifosfamide in a 6-organoid System 92 Figure 3.1. Patient-derived models for personalized precision medicine 111 Figure 3.2. Experimental methodology flow chart for patient-derived tumor Models 119 Figure 3.3. PDO in a Tumor-on-a-chip system 120 Figure 3.4 HNSCC Patient tumor construct histological assessment 130 vi Figure 3.5 In vitro drug response comparison of head and neck tumor between sponge assay and Patient-derived Organoid 133 Figure 3.6. Normalized and consolidated ATP assay viability data of 8 head and neck cancer patients 135 Figure 3.7 Inhibition effect by dicoumarol on β-lap cytotoxicity 137 Figure 3.8. Maximum inhibition effect of Dicoumorol of β-Lap induced toxicity tested on radiation insensitive rSCC61 and radiation sensitive SCC61 cells 3D tumor organoid 140 Figure 3.9. Mesothelioma patient tumor construct histological assessment 143 Figure 3.10. In vitro chemotherapy assessment in organoids derived from mesothelioma patients 147 Figure 4.1. Device and construct fabrication 171 Figure 4.2. Interstitial flow effects 178 Figure 4.3. Effect of hydrogel matrix stiffness on metastatic migration of HCT116 cells in 3D 181 Figure 4.4. Effects of drugs on in vitro HCT-116 invasion 184 Figure 4.5. HCT-116 invasion quantification for all conditions and all time points 185 Figure 4.6. HCT-116 viability under 5FU insult 186 Figure 4.7. HCT-116 viability following Marimastat insult 189 Figure 4.8. Dissolving of PEGSSDA based hydrogel 191 Figure 4.9. Isolating migrated cells in Co-culture of mCherry HCT116 & Caco-2 193 Figure 5.1 Mesothelioma Patient tumor construct invasion dynamics and drug Sensitivity 215 vii Figure 5.2 GBM Patient tumor construct histological assessment 217 Table 3.1 Patient tumor characteristics and treatment history along with the administered drug to respective PDOs 127 APPENDIX: LIST OF FIGURES AND TABLES A 1. 1. Lung-on-a-chip device fabrication. 245 A 1. 2. Schematic of lung organoid layers 252 A 1.3. Lung-on-a-chip experimental set-up 254 A 1. 4. Adapter for the Lung-on-a-Chip 255 A 1. 5. Lung-on-a-chip results 256 A 2. 1. Patient 1 organoid LIVE/DEAD segmentation 257 A 2. 2. Patient 2 organoid LIVE/DEAD segmentation 258 A 3. 1. Schematic of bubble trap 259 A 3. 2. Interstitial flow influence on migrated cells 260 A 3. 3 Expanded view of the typical maximum projection confocal micrographs of cell invasion for all 5FU conditions and time points 261 A 3. 4. Expanded view of the typical maximum projection confocal micrographs of cell invasion for all Marimastat conditions and time points 262 A 3. 5. LIVE/DEAD viability of HCT-116 under 5FU insult 263 A 3. 6. LIVE/DEAD viability of HCT-116 under Marimastat insult 264 viii LIST OF ABBREVIATIONS 2D Two-dimensional 3D Three-dimensional 5’-DFCR 5'-deoxy-S-fluorocytidine 5'-DFUR 5'-deoxy-S-fluorouridine 5-FU 5-fluorouracil A549 Adenocarcinoma human alveolar basal epithelial cell AFB Adhesive film-based ALI medium Air-liquid interface medium Alpha MEM Minimum Essential Medium Eagle- Alpha Modification ANOVA Analysis of variance ATP Adenosine triphosphate BDNF Brain-derived neurotrophic factor BEGM Bronchial epithelial cell growth B-Lap B-Lapachone BOC Body-on-a-chip cAMP Cyclic adenosine monophosphate CCM Cardiomyocyte culture media CE Cardiac endothelium cells Cis Cisplatin CM Cardiomyocytes CNC Computer numerical control COC/COP Cyclo-olefin (co)polymer CRC Colorectal cancer cells CRS Cytoreductive surgery CT Computerized tomography CTO Cell line-based Tumor organoid DAPI 4′,6-diamidino-2-phenylindole DI water Deionized water DIC Dicoumorol ix DMEM Dulbecco’s Modified Eagle’s Media DMEM/F-12 Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 DMSO Dimethylsulfoxide DNA Deoxyribonucleic acid DST Double-sided tape ECFR-P Phosphorylated epidermal growth factor receptor ECM Extracellular matrix EDTA Ethylenediaminetetraacetic acid EGF Epidermal growth factor EGFR Epidermal growth factor receptor EGM Endothelial cell growth media ELISA Enzyme-linked immunosorbent assay EO Ethylene Oxide sterilization FBS Fetal bovine serum FDA Federal Drug Administration GAG Glycosaminoglycan GBM Glioblastoma multiforme GI Gastro-Intestinal HA Human astrocytes cells HA Hyaluronic acid HBMEC Human brain microvascular endothelial cells HBVP Human brain vascular pericytes HCM Hepatocyte culture media HCT116 Human colon carcinoma cells HDRA Histoculture drug response assay HEMA Hydroxyethyl methacrylate HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid HH Human hepatocytes HIPEC Heated intraperitoneal chemotherapy HLEc Human liver-derived endothelial cells HM Human iPSC-derived microglia x HN Human neural cells HNSCC Head and neck squamous cell carcinoma HO Human oligodendrocytes