Cell Assembly with 3D Bioprinting

Provides an up-to-date outline of cell assembly methods and applications of 3D bioprinting Cell Assembly with 3D Bioprinting provides an accesible overview of the layer-by-layer manufacturing of living structures using biomaterials. Focusing on technical implemention in medical and bioengineering applications, this practical guide summarize each key aspect of the 3D bioprinting process. Contributions from a team of leading researchers describe bioink preparation, printing method selection, experimental protocols, integration with specific applications, and more.  Detailed, highly illustrated chapters cover different bioprinting approaches and their applications, including coaxial bioprinting, digital light projection, direct ink writing, liquid support bath-assisted 3D printing, and microgel-, microfiber-, and microfluidics-based biofabrication. The book includes practical examples of 3D bioprinting, a protocol for typical 3D bioprinting, and relevant experimental data drawn from recent research. * Highlights the interdisciplinary nature of 3D bioprinting and its applications in biology, medicine, and pharmaceutical science * Summarizes a variety of commonly used 3D bioprinting methods * Describes the design and preparation of various types of bioinks  * Discusses applications of 3D bioprinting such as organ development, toxicological research, clinical transplantation, and tissue repair Covering a wide range of topics, Cell Assembly with 3D Bioprinting is essential reading for advanced students, academic researchers, and industry professionals in fields including biomedicine, tissue engineering, bioengineering, drug development, pharmacology, bioglogical screening, and mechanical engineering.

Table of Contents:
Preface xv 1 3D Bioprinting, A Powerful Tool for 3D Cells Assembling 1 1.1 What Is 3D Bioprinting? 1 1.2 Evolution of 3D Bioprinting 3 1.3 Brief Classification of 3D Bioprinting 4 1.4 Evaluation of Bioinks 5 1.5 Outlook and Discussion 6 References 8 2 Representative 3D Bioprinting Approaches 11 2.1 Introduction 11 2.2 Inkjet Bioprinting 13 2.2.1 Mechanisms of Droplet Formation 14 2.2.1.1 Continuous-Inkjet Bioprinting 14 2.2.1.2 Drop-on-Demand Inkjet Bioprinting 15 2.2.1.3 Electrohydrodynamic Jet Bioprinting 16 2.2.2 Hydrogel-Based Bioinks for Inkjet Bioprinting 17 2.2.2.1 Material Properties for Inkjet Bioprinting Applications 18 2.2.2.2 Commonly Used Hydrogels in Inkjet Bioprinting 19 2.2.3 Representative Cell Printing Applications 20 2.2.3.1 Bone and Cartilage Tissues 21 2.2.3.2 Organoids 22 2.2.3.3 Skin Tissues 22 2.2.3.4 Vascular Networks 22 2.2.4 Summary 22 2.3 Extrusion Bioprinting 23 2.3.1 Mechanisms of Extruding Biocompatible Materials 23 2.3.2 Primary Extrusion Bioprinting Strategies 24 2.3.3 Main Categories of Extrudable Biomaterials 25 2.3.3.1 Hydrogels 25 2.3.3.2 Micro-Carriers 26 2.3.3.3 Cell Aggregates 27 2.3.3.4 Decellularized Matrix Components 28 2.3.4 Summary 28 2.4 Light-Based Bioprinting 28 2.4.1 Laser-Assisted Bioprinting 28 2.4.1.1 Mechanism 28 2.4.1.2 Materials 30 2.4.1.3 Biomedical Applications 30 2.4.2 Stereolithography 32 2.4.2.1 Mechanism 32 2.4.2.2 Materials 33 2.4.2.3 Biomedical Applications 33 2.4.3 Multi-Photon Polymerization 34 2.4.3.1 Mechanism 34 2.4.3.2 Materials 35 2.4.3.3 Biomedical Applications 35 2.4.4 Digital Light Projection 3D Printing 35 2.4.4.1 Mechanism 36 2.4.4.2 Materials 37 2.4.4.3 Biomedical Applications 37 2.4.5 Computed Axial Lithography 37 2.4.5.1 Mechanism 37 2.4.5.2 Materials and Biomedical Applications 37 2.4.6 Summary 38 References 38 3 Bioink Design: From Shape to Function 47 3.1 Significance of Bioink Design 47 3.2 Categories of Bioink 47 3.3 Three Evaluation Criteria of Bioink 48 3.3.1 Printability 48 3.3.2 Mechanical Properties 48 3.3.3 Biocompatibility 48 3.4 Strategies for Enabling the Printability 49 3.4.1 Optimization of Cross-linking Sequence 49 3.4.2 Support Material-Assisted Bioprinting 50 3.4.3 Microgel-Based Bioink 50 3.5 Strategies for Bioink Reinforcement 50 3.5.1 Composite Bioink Design 50 3.5.2 Microfiber-Assisted Reinforcement 51 3.6 Strategies for Improving the Biocompatibility 51 3.7 Representative Bioink Design Case: GelMA-Based Bioinks 52 3.7.1 Property Characterization of the GelMA Bioink 52 3.7.2 3D Bioprinting of GelMA Bioinks with Dual Cross-linking Strategy 53 3.7.3 3D Bioprinting of GelMA Bioinks with Nanoclay as Support 55 3.8 Commercial Bioink 57 3.8.1 GelMA (EFL-GM Series) 58 3.8.2 Fluorescent GelMA (EFL-GM-F Series) 58 3.8.3 Porous GelMA (EFL-GM-PR Series) 60 3.8.4 HAMA (EFL-HAMA Series) 64 3.8.5 SilMA (EFL-SilMA Series) 64 3.8.6 PCLMA (EFL-PCLMA Series) 64 References 66 4 Coaxial 3D Bioprinting 69 4.1 Introduction 69 4.1.1 Significance 69 4.1.2 Two Categories 72 4.1.2.1 Solid Fiber-Based Coaxial Bioprinting 72 4.1.2.2 Hollow Fiber-Based Coaxial Bioprinting 73 4.2 Printable Ink Materials 74 4.2.1 Forming Mechanism 74 4.2.2 Categories of Printable Bioinks 75 4.2.2.1 Alginate 75 4.2.2.2 Gelatin 78 4.2.2.3 GelMA 79 4.3 Representative Biomedical Applications 80 4.3.1 Morphology-Controllable Microfiber-Based Organoids 80 4.3.2 Vessel-on-a-Chip 81 4.4 Future Perspective 85 References 86 5 Digital Light Projection-Based 3D Bioprinting 89 5.1 Introduction 89 5.1.1 Printing Process 89 5.1.2 Significance 89 5.2 Photocurable Biomaterials 91 5.2.1 Photo-Cross-Linking Mechanism 92 5.2.1.1 Conversion of Light Energy to Chemical Energy: Photoinitiator 92 5.2.1.2 Formation of Molecular Network: Monomer Polymerization 93 5.2.2 Typical Materials: Gelatin Methacryloyl (GelMA) 94 5.2.2.1 Composition and Synthesis 94 5.2.2.2 Substitution Degree 95 5.3 Printing Equipment 96 5.3.1 Optical Units 96 5.3.1.1 Image Forming: Digital Micromirror Devices 97 5.3.1.2 Objective Lens: Focusing System 97 5.3.1.3 Material Storage Units 98 5.3.1.4 Environment Controlling Systems 98 5.3.1.5 Ink Tank: Transparent and Non-stick Bottom 99 5.4 Mechanical Movement Units 99 5.4.1 Lifting Mechanism: Main Movement 99 5.4.2 Tilting Mechanism: Mixing and Separation 100 5.4.2.1 Printing Error Formation and Optimization Strategies 100 5.5 Optimization of Several Typical Structures 102 5.5.1 Printing Strategies of Solid Structures 103 5.5.2 Printing Strategies of Channel Structures 104 5.5.3 Printing Strategies of Conduit Structures 104 5.5.4 Printing Strategies of Thin-Walled Structures 105 5.5.5 Printing Strategies of Microcolumn Structures 105 5.6 Applications 107 5.6.1 DLPBP Structures with High Precision 107 5.6.2 Customized Physical Properties Bioprinting 107 5.6.3 Regenerative and Biomedical Applications 108 References 110 6 Direct Ink Writing for 3D Bioprinting Applications 113 6.1 Introduction 113 6.2 Printable Bioinks in DIW 114 6.2.1 Supporting Mechanisms and Representative Bioinks 115 6.2.1.1 Rapid Solidification-Induced Mechanical Stiffness Improvement 115 6.2.1.2 Yield-stress Additive-Induced Self-Supporting Capacity 119 6.2.2 Design Criteria of Bioinks for Direct Writing Applications 121 6.2.2.1 Rheological Properties 122 6.2.2.2 Cross-linking Capacity 122 6.2.2.3 Biocompatibility and Biodegradation 123 6.2.2.4 Mechanical Properties 124 6.3 Technical Specifics in Direct Ink Writing 124 6.3.1 Investigation on Printability of Bioinks 124 6.3.2 Different Printing Strategies in Rapid Solidification-Induced 3D Printing Approach 126 6.3.2.1 Printing of Thermal Cross-linkable Biomaterials 126 6.3.2.2 Printing of Ionic Cross-linkable Biomaterials 127 6.3.2.3 Printing of Photo Cross-linkable Biomaterials 128 6.3.2.4 Printing of Enzyme Cross-linkable Biomaterials 129 6.3.3 3D Structure Printing Using Self-Supporting Material-Assisted 3D Printing Approach 130 6.3.3.1 Internal Scaffold Additive-Assisted 3D Printing 130 6.3.3.2 Microgel Additive-Assisted 3D Printing 132 6.4 Representative Biomedical Applications 132 6.4.1 Aortic Valve Printing 132 6.4.2 Bone and Cartilage Tissue Printing 133 6.4.3 Cardiac Tissue Printing 134 6.4.4 Liver Tissue Printing 135 6.4.5 Lung Tissue Printing 135 6.4.6 Neural Tissue Printing 135 6.4.7 Eye and Ear Printing 136 6.4.8 Pancreas Printing 137 6.4.9 Skin Tissue Printing 137 6.4.10 Blood Vessel Printing 138 6.5 Conclusions and Future Work 138 References 139 7 Liquid Support Bath–Assisted 3D Bioprinting 149 7.1 Introduction 149 7.2 Liquid Support Bath Materials 150 7.2.1 Support Bath Materials Based on Different Supporting Mechanisms 151 7.2.1.1 Unrecoverable Matrix Materials 151 7.2.1.2 Buoyant Support Fluids 151 7.2.1.3 Reversibly Self-Healing Hydrogels 153 7.2.1.4 Yield-Stress Fluids 154 7.2.2 Preparation Methods 156 7.2.2.1 Microparticle Aggregation 156 7.2.2.2 Homogenous Suspensions with Micro/Nanostructures 157 7.2.2.3 Chemical Synthesis 158 7.2.2.4 Other Methods 158 7.2.3 Design Criteria for Ideal Liquid Support Bath Material 158 7.2.3.1 Rheological Properties 158 7.2.3.2 Chemical Stability 159 7.2.3.3 Physical Stability 159 7.2.3.4 Biocompatibility 161 7.2.3.5 Hydrophilicity and Hydrophobicity 161 7.2.3.6 Others 161 7.3 Scientific Issues During Liquid Support Bath–Assisted 3D Printing 162 7.3.1 Effects of Operating Conditions on Filament Formation in Support Bath 162 7.3.2 Effects of Support Bath Materials on Filament Morphology 162 7.3.2.1 Rheological Properties of Support Bath Materials 162 7.3.2.2 Diffusion of Ink Materials into Surrounding Support Bath 163 7.3.2.3 Interfacial Tension–Induced Filament Deformation 165 7.3.3 Effects of Nozzle Movement on the Printed Structure 165 7.3.4 Path Design in Liquid Support Bath–Assisted 3D Printing 166 7.4 Post-treatments for Liquid Support Bath–Assisted 3D Printing 167 7.4.1 Post-treatments in e-3DP 167 7.4.2 Post-treatments in Support Bath–Enabled 3D Printing 169 7.5 Representative Biomedical Applications 169 7.5.1 Organ Printing 169 7.5.2 Lab-on-a-Chip 171 7.5.3 Other Bio-Related Applications 173 7.6 Conclusions and Future Directions 173 References 175 8 Bioprinting Approaches of Hydrogel Microgel 179 8.1 Introduction 179 8.2 Auxiliary Dripping 179 8.2.1 Inkjet Printing 180 8.2.1.1 Piezoelectric Inkjet 180 8.2.1.2 Thermal Bubble Inkjet 183 8.2.2 Laser-Assisted Printing 184 8.2.3 Electrohydrodynamic Printing 185 8.3 Diphase Emulsion 195 8.3.1 Nonaqueous Liquid Stirring 195 8.3.2 Air-Assisted Atomization 197 8.3.3 Microfluidic Technology 198 8.4 Lithography Technology 202 8.4.1 Replica Mold 202 8.4.2 Discrepant Wettability 203 8.4.3 Photomask Film 206 8.4.4 Digital Light Processing 208 8.5 Bulk Crushing 208 References 211 9 Biomedical Applications of Microgels 213 9.1 Introduction 213 9.1.1 Tiny Size 213 9.1.2 Hydrogel Network 213 9.1.3 Complex Mechanical Properties 214 9.2 In Vitro Model 214 9.3 Cell Therapy 216 9.4 Drug Delivery 219 9.5 Cell Amplification 223 9.6 Single-Cell Capture 227 9.7 Supporting Matrices 229 9.8 Secondary Bioprinting 232 References 235 10 Microfiber-Based Organoids Bioprinting for In Vitro Model 237 10.1 Introduction 237 10.1.1 Significance and Challenge 237 10.1.2 Hydrogel Materials 238 10.2 Coaxial Bioprinting of Bioactive Cell-laden Microfiber 238 10.2.1 Microfluidic Coaxial Bioprinting 239 10.2.2 Coaxial Nozzle-Assisted Bioprinting 240 10.3 Heteromorphic/Heterogeneous Microfiber Bioprinting 241 10.3.1 Heteromorphic Microfiber 242 10.3.2 Heterogeneous Microfiber 244 10.4 3D Assembly of Microfibers 245 10.4.1 3D Bioweaving 245 10.4.2 3D Bioprinting 245 10.5 Microfiber-Based Organoids Bioprinting for In Vitro Mini Tissue Models 247 10.5.1 Vascular Organoid 247 10.5.2 Myocyte Fiber 248 10.5.3 Nerve Fiber 248 10.5.4 Cardiomyocyte Fiber 249 10.5.5 Co-cultured Multi-organoids Interactions 249 10.6 Discussion and Outlook 250 References 251 11 Large Scale Tissues Bioprinting 257 11.1 Introduction 257 11.1.1 Challenges in Bioprinting Large Scale Tissues 257 11.1.2 Strategies in Bioprinting Large Scale Tissue with Nutrient Networks 258 11.1.2.1 Porous Network Printing 258 11.1.2.2 Hollow Channel Network Printing 259 11.1.2.3 Advanced Bioprinting Techniques-Enabled Printing Highly Biomimetic Vascular Network 259 11.2 Large Scale Cell-laden Porous Structures Printing 259 11.2.1 Independent Porous Structure Printing 259 11.2.2 Interconnected Porous Structure Printing 261 11.2.2.1 Directly Cell-laden Scaffold Printing 261 11.2.2.2 Synchronous Bioprinting (Bioink and Sacrificial Ink Half and Half) 261 11.2.3 Heterogeneous Independent/Interconnected Porous Structure Printing 262 11.2.4 Long-term Perfusion Culture on a Chip 265 11.2.5 Discussions (Properties, Pros, Cons, etc.) 265 11.3 Large Scale Cell-laden Structures with Vascular Channel Printing 266 11.3.1 Sacrificial Bioprinting 266 11.3.2 Coaxial Bioprinting 267 11.4 One-step Coaxial/Sacrificial Printing of Large Scale Vascularized Tissue Constructs 268 11.4.1 Mechanism 268 11.4.2 Freeform Structure with Vascular Channels Printing 269 11.4.3 Heterogeneous Structure with Vascular Channels Printing 270 11.4.4 Long-term Perfusion Culture on a Chip 272 11.4.5 Discussion (Properties, Pros and Cons, etc.) 272 11.5 Advanced Bioprinting Technique-Enabled Printing Highly Biomimetic Tissues 273 11.5.1 Support Bath-Assisted Bioprinting 273 11.5.2 Light-Based Bioprinting 273 11.5.3 Discussion (Properties, Pros and Cons, etc.) 275 11.6 Representative Biomedical Applications 275 References 276 12 3D Printing of Vascular Chips 281 12.1 Introduction 281 12.2 Construction Process of Hydrogel-Based Vascular Chips 282 12.2.1 Damage-Free Demolding Process Based on Soft Fiber Template 282 12.2.1.1 Damage-Free Demolding Process 283 12.2.1.2 Comparative Analysis of Damage-Free and Conventional Demolding Processes 283 12.2.2 Hydrogel Bonding Strategy Based on Twice-Cross-linking Mechanism 286 12.2.2.1 Manufacturing Process of Hydrogel-Based Microfluidic Chips 287 12.2.2.2 Mechanism Study 287 12.2.2.3 Material Selection 288 12.2.2.4 Feasible Domain 289 12.2.2.5 Bonding Results 289 12.2.3 Multi-Scale 3D Printing Process 291 12.2.3.1 Mechanism of Multi-Scale 3D Printing Process 291 12.2.3.2 Printing Parameters 292 12.2.4 Construction Process of Hydrogel-Based Vascular Chips 293 12.3 Characterization of Vascular Chips 295 12.3.1 Fundamental Characterization of Vascular Chips 295 12.3.1.1 Characterization of Endothelium Function of Channels 295 12.3.1.2 Characterization of Endothelial Cells Viability 295 12.3.1.3 Characterization of Endothelial Cells Morphology 296 12.3.1.4 Characterization of Endothelium Channel 297 12.3.2 Morphology Characterization of Hydrogel-Based Vascular Chips 298 12.3.2.1 Multi-Level Bifurcated Channel Network Structure 298 12.3.2.2 Multi-Scale Vascular Model 299 12.3.2.3 Biomimicking Vascular Model 299 12.3.3 Characterization of Vascular Function 302 12.3.3.1 Nutrition Supply Function 302 12.3.3.2 Expression of Key Functional Proteins in Endothelial Cells 302 12.3.3.3 Simulation of Vascular Inflammation Reaction 303 12.3.3.4 Characterization of Vascular Barrier Function 304 12.4 Conclusion 307 References 308 13 3D Printing of In Vitro Models 311 13.1 Introduction 311 13.2 Typical 3D Bioprinting Technologies and Common Target Tissue/Organ Demand 312 13.2.1 Inkjet-Based Bioprinting 313 13.2.2 Extrusion-Based Bioprinting 314 13.2.3 Light-Assisted Bioprinting 315 13.3 Developing Process of In Vitro Models 316 13.3.1 Mini-Tissue in 3D Growth State 316 13.3.1.1 Sphere Mini-Tissue Model 316 13.3.1.2 Fiber Mini-Tissue Model 317 13.3.1.3 Array Mini-Tissue Model 318 13.3.1.4 Limitations 319 13.3.2 Organ-on-a-Chip with Multiplex Microenvironment 319 13.3.2.1 Integrated Organ-on-a-Chip 321 13.3.2.2 Modular Microfluidic System 322 13.3.2.3 Multiple-Organ System 323 13.3.2.4 Limitations 325 13.3.3 Tissue/Organ Construct with Biomimicking Property 325 13.3.3.1 Vascular Construct 326 13.3.3.2 Vascularized Tissue Construct 328 13.3.3.3 Limitations 330 13.4 3D Printing of In Vitro Tumor Models 330 13.4.1 Tumor Cell-Laden Construct 330 13.4.2 Multi-Cell Tumor Sphere 331 13.4.3 Tumor Metastasis Model with Angiogenesis 332 13.5 Summary and Prospect 334 13.5.1 Key Virtue and Comparison 334 13.5.2 Outlook 334 13.5.2.1 3D Bioprinting Technology 335 13.5.2.2 Individual Differences 335 13.5.2.3 Systematic Interaction 335 13.5.2.4 Industrialization 335 13.6 Conclusions 336 References 336 14 Protocol of Typical 3D Bioprinting 339 Reference 343 Index 345

About the Author :
Yong He obtained his PhD degree in mechanical engineering at the ZheJiang University in 2008. He is currently a professor at College of Mechanical Engineering, ZheJiang University, China. He is also the deputy director of Key Lab of 3D Printing Process and Equipment of ZheJiang Province. His research is focused on the biofabrication with 3D printing especially on the building organs on chips. He has published more than 100 international journal papers and authorized over 30 patents. He has developed many special 3D printers for the fabrication of microfluidic devices, such as 3D sugar printer and 3D softmatter printer. Qing Gao obtained his BSc in mechanical design, manufacturing and automation at Hefei University of Technology in 2012. In 2017 he obtained his PhD degree in mechanical manufacturing and automation at the ZheJiang University and continue working in the university as a postdoc. He engages in research on biomanufacturing, biological 3D printing, organ chips, etc. As a core member, he has developed a portable biological 3D printer and high-performance GelMA bio-ink and is committed to building a "material + equipment + service" integrated intelligent manufacturing product system. Yifei Jin received his Ph.D. in mechanical engineering from the University of Florida in 2018, He joined the Department of Mechanical Engineering at the University of Nevada, ¿Reno as assistant professor in July 2019. His primary research interests mainly involve 3D bioprinting of living tissue constructs, 3D printing of hydrophobic functional materials, yield-stress fluids for 3D printing applications, stimuli-responsive materials for 4D printing applications, and fabrication of multi-layered capsules. His research emphasizes the coupling of materials and fabrication approaches to develop novel 3D printing techniques and understand the underlying physics during printing.