Cell Assembly with 3D Bioprinting

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.

• Preface xv1 3D Bioprinting, A Powerful Tool for 3D Cells Assembling 11.1 What Is 3D Bioprinting? 11.2 Evolution of 3D Bioprinting 31.3 Brief Classification of 3D Bioprinting 41.4 Evaluation of Bioinks 51.5 Outlook and Discussion 6References 82 Representative 3D Bioprinting Approaches 112.1 Introduction 112.2 Inkjet Bioprinting 132.2.1 Mechanisms of Droplet Formation 142.2.1.1 Continuous-Inkjet Bioprinting 142.2.1.2 Drop-on-Demand Inkjet Bioprinting 152.2.1.3 Electrohydrodynamic Jet Bioprinting 162.2.2 Hydrogel-Based Bioinks for Inkjet Bioprinting 172.2.2.1 Material Properties for Inkjet Bioprinting Applications 182.2.2.2 Commonly Used Hydrogels in Inkjet Bioprinting 192.2.3 Representative Cell Printing Applications 202.2.3.1 Bone and Cartilage Tissues 212.2.3.2 Organoids 222.2.3.3 Skin Tissues 222.2.3.4 Vascular Networks 222.2.4 Summary 222.3 Extrusion Bioprinting 232.3.1 Mechanisms of Extruding Biocompatible Materials 232.3.2 Primary Extrusion Bioprinting Strategies 242.3.3 Main Categories of Extrudable Biomaterials 252.3.3.1 Hydrogels 252.3.3.2 Micro-Carriers 262.3.3.3 Cell Aggregates 272.3.3.4 Decellularized Matrix Components 282.3.4 Summary 282.4 Light-Based Bioprinting 282.4.1 Laser-Assisted Bioprinting 282.4.1.1 Mechanism 282.4.1.2 Materials 302.4.1.3 Biomedical Applications 302.4.2 Stereolithography 322.4.2.1 Mechanism 322.4.2.2 Materials 332.4.2.3 Biomedical Applications 332.4.3 Multi-Photon Polymerization 342.4.3.1 Mechanism 342.4.3.2 Materials 352.4.3.3 Biomedical Applications 352.4.4 Digital Light Projection 3D Printing 352.4.4.1 Mechanism 362.4.4.2 Materials 372.4.4.3 Biomedical Applications 372.4.5 Computed Axial Lithography 372.4.5.1 Mechanism 372.4.5.2 Materials and Biomedical Applications 372.4.6 Summary 38References 383 Bioink Design: From Shape to Function 473.1 Significance of Bioink Design 473.2 Categories of Bioink 473.3 Three Evaluation Criteria of Bioink 483.3.1 Printability 483.3.2 Mechanical Properties 483.3.3 Biocompatibility 483.4 Strategies for Enabling the Printability 493.4.1 Optimization of Cross-linking Sequence 493.4.2 Support Material-Assisted Bioprinting 503.4.3 Microgel-Based Bioink 503.5 Strategies for Bioink Reinforcement 503.5.1 Composite Bioink Design 503.5.2 Microfiber-Assisted Reinforcement 513.6 Strategies for Improving the Biocompatibility 513.7 Representative Bioink Design Case: GelMA-Based Bioinks 523.7.1 Property Characterization of the GelMA Bioink 523.7.2 3D Bioprinting of GelMA Bioinks with Dual Cross-linking Strategy 533.7.3 3D Bioprinting of GelMA Bioinks with Nanoclay as Support 553.8 Commercial Bioink 573.8.1 GelMA (EFL-GM Series) 583.8.2 Fluorescent GelMA (EFL-GM-F Series) 583.8.3 Porous GelMA (EFL-GM-PR Series) 603.8.4 HAMA (EFL-HAMA Series) 643.8.5 SilMA (EFL-SilMA Series) 643.8.6 PCLMA (EFL-PCLMA Series) 64References 664 Coaxial 3D Bioprinting 694.1 Introduction 694.1.1 Significance 694.1.2 Two Categories 724.1.2.1 Solid Fiber-Based Coaxial Bioprinting 724.1.2.2 Hollow Fiber-Based Coaxial Bioprinting 734.2 Printable Ink Materials 744.2.1 Forming Mechanism 744.2.2 Categories of Printable Bioinks 754.2.2.1 Alginate 754.2.2.2 Gelatin 784.2.2.3 GelMA 794.3 Representative Biomedical Applications 804.3.1 Morphology-Controllable Microfiber-Based Organoids 804.3.2 Vessel-on-a-Chip 814.4 Future Perspective 85References 865 Digital Light Projection-Based 3D Bioprinting 895.1 Introduction 895.1.1 Printing Process 895.1.2 Significance 895.2 Photocurable Biomaterials 915.2.1 Photo-Cross-Linking Mechanism 925.2.1.1 Conversion of Light Energy to Chemical Energy: Photoinitiator 925.2.1.2 Formation of Molecular Network: Monomer Polymerization 935.2.2 Typical Materials: Gelatin Methacryloyl (GelMA) 945.2.2.1 Composition and Synthesis 945.2.2.2 Substitution Degree 955.3 Printing Equipment 965.3.1 Optical Units 965.3.1.1 Image Forming: Digital Micromirror Devices 975.3.1.2 Objective Lens: Focusing System 975.3.1.3 Material Storage Units 985.3.1.4 Environment Controlling Systems 985.3.1.5 Ink Tank: Transparent and Non-stick Bottom 995.4 Mechanical Movement Units 995.4.1 Lifting Mechanism: Main Movement 995.4.2 Tilting Mechanism: Mixing and Separation 1005.4.2.1 Printing Error Formation and Optimization Strategies 1005.5 Optimization of Several Typical Structures 1025.5.1 Printing Strategies of Solid Structures 1035.5.2 Printing Strategies of Channel Structures 1045.5.3 Printing Strategies of Conduit Structures 1045.5.4 Printing Strategies of Thin-Walled Structures 1055.5.5 Printing Strategies of Microcolumn Structures 1055.6 Applications 1075.6.1 DLPBP Structures with High Precision 1075.6.2 Customized Physical Properties Bioprinting 1075.6.3 Regenerative and Biomedical Applications 108References 1106 Direct Ink Writing for 3D Bioprinting Applications 1136.1 Introduction 1136.2 Printable Bioinks in DIW 1146.2.1 Supporting Mechanisms and Representative Bioinks 1156.2.1.1 Rapid Solidification-Induced Mechanical Stiffness Improvement 1156.2.1.2 Yield-stress Additive-Induced Self-Supporting Capacity 1196.2.2 Design Criteria of Bioinks for Direct Writing Applications 1216.2.2.1 Rheological Properties 1226.2.2.2 Cross-linking Capacity 1226.2.2.3 Biocompatibility and Biodegradation 1236.2.2.4 Mechanical Properties 1246.3 Technical Specifics in Direct Ink Writing 1246.3.1 Investigation on Printability of Bioinks 1246.3.2 Different Printing Strategies in Rapid Solidification-Induced 3D Printing Approach 1266.3.2.1 Printing of Thermal Cross-linkable Biomaterials 1266.3.2.2 Printing of Ionic Cross-linkable Biomaterials 1276.3.2.3 Printing of Photo Cross-linkable Biomaterials 1286.3.2.4 Printing of Enzyme Cross-linkable Biomaterials 1296.3.3 3D Structure Printing Using Self-Supporting Material-Assisted 3D Printing Approach 1306.3.3.1 Internal Scaffold Additive-Assisted 3D Printing 1306.3.3.2 Microgel Additive-Assisted 3D Printing 1326.4 Representative Biomedical Applications 1326.4.1 Aortic Valve Printing 1326.4.2 Bone and Cartilage Tissue Printing 1336.4.3 Cardiac Tissue Printing 1346.4.4 Liver Tissue Printing 1356.4.5 Lung Tissue Printing 1356.4.6 Neural Tissue Printing 1356.4.7 Eye and Ear Printing 1366.4.8 Pancreas Printing 1376.4.9 Skin Tissue Printing 1376.4.10 Blood Vessel Printing 1386.5 Conclusions and Future Work 138References 1397 Liquid Support Bath–Assisted 3D Bioprinting 1497.1 Introduction 1497.2 Liquid Support Bath Materials 1507.2.1 Support Bath Materials Based on Different Supporting Mechanisms 1517.2.1.1 Unrecoverable Matrix Materials 1517.2.1.2 Buoyant Support Fluids 1517.2.1.3 Reversibly Self-Healing Hydrogels 1537.2.1.4 Yield-Stress Fluids 1547.2.2 Preparation Methods 1567.2.2.1 Microparticle Aggregation 1567.2.2.2 Homogenous Suspensions with Micro/Nanostructures 1577.2.2.3 Chemical Synthesis 1587.2.2.4 Other Methods 1587.2.3 Design Criteria for Ideal Liquid Support Bath Material 1587.2.3.1 Rheological Properties 1587.2.3.2 Chemical Stability 1597.2.3.3 Physical Stability 1597.2.3.4 Biocompatibility 1617.2.3.5 Hydrophilicity and Hydrophobicity 1617.2.3.6 Others 1617.3 Scientific Issues During Liquid Support Bath–Assisted 3D Printing 1627.3.1 Effects of Operating Conditions on Filament Formation in Support Bath 1627.3.2 Effects of Support Bath Materials on Filament Morphology 1627.3.2.1 Rheological Properties of Support Bath Materials 1627.3.2.2 Diffusion of Ink Materials into Surrounding Support Bath 1637.3.2.3 Interfacial Tension–Induced Filament Deformation 1657.3.3 Effects of Nozzle Movement on the Printed Structure 1657.3.4 Path Design in Liquid Support Bath–Assisted 3D Printing 1667.4 Post-treatments for Liquid Support Bath–Assisted 3D Printing 1677.4.1 Post-treatments in e-3DP 1677.4.2 Post-treatments in Support Bath–Enabled 3D Printing 1697.5 Representative Biomedical Applications 1697.5.1 Organ Printing 1697.5.2 Lab-on-a-Chip 1717.5.3 Other Bio-Related Applications 1737.6 Conclusions and Future Directions 173References 1758 Bioprinting Approaches of Hydrogel Microgel 1798.1 Introduction 1798.2 Auxiliary Dripping 1798.2.1 Inkjet Printing 1808.2.1.1 Piezoelectric Inkjet 1808.2.1.2 Thermal Bubble Inkjet 1838.2.2 Laser-Assisted Printing 1848.2.3 Electrohydrodynamic Printing 1858.3 Diphase Emulsion 1958.3.1 Nonaqueous Liquid Stirring 1958.3.2 Air-Assisted Atomization 1978.3.3 Microfluidic Technology 1988.4 Lithography Technology 2028.4.1 Replica Mold 2028.4.2 Discrepant Wettability 2038.4.3 Photomask Film 2068.4.4 Digital Light Processing 2088.5 Bulk Crushing 208References 2119 Biomedical Applications of Microgels 2139.1 Introduction 2139.1.1 Tiny Size 2139.1.2 Hydrogel Network 2139.1.3 Complex Mechanical Properties 2149.2 In Vitro Model 2149.3 Cell Therapy 2169.4 Drug Delivery 2199.5 Cell Amplification 2239.6 Single-Cell Capture 2279.7 Supporting Matrices 2299.8 Secondary Bioprinting 232References 23510 Microfiber-Based Organoids Bioprinting for In Vitro Model 23710.1 Introduction 23710.1.1 Significance and Challenge 23710.1.2 Hydrogel Materials 23810.2 Coaxial Bioprinting of Bioactive Cell-laden Microfiber 23810.2.1 Microfluidic Coaxial Bioprinting 23910.2.2 Coaxial Nozzle-Assisted Bioprinting 24010.3 Heteromorphic/Heterogeneous Microfiber Bioprinting 24110.3.1 Heteromorphic Microfiber 24210.3.2 Heterogeneous Microfiber 24410.4 3D Assembly of Microfibers 24510.4.1 3D Bioweaving 24510.4.2 3D Bioprinting 24510.5 Microfiber-Based Organoids Bioprinting for In Vitro Mini Tissue Models 24710.5.1 Vascular Organoid 24710.5.2 Myocyte Fiber 24810.5.3 Nerve Fiber 24810.5.4 Cardiomyocyte Fiber 24910.5.5 Co-cultured Multi-organoids Interactions 24910.6 Discussion and Outlook 250References 25111 Large Scale Tissues Bioprinting 25711.1 Introduction 25711.1.1 Challenges in Bioprinting Large Scale Tissues 25711.1.2 Strategies in Bioprinting Large Scale Tissue with Nutrient Networks 25811.1.2.1 Porous Network Printing 25811.1.2.2 Hollow Channel Network Printing 25911.1.2.3 Advanced Bioprinting Techniques-Enabled Printing Highly Biomimetic Vascular Network 25911.2 Large Scale Cell-laden Porous Structures Printing 25911.2.1 Independent Porous Structure Printing 25911.2.2 Interconnected Porous Structure Printing 26111.2.2.1 Directly Cell-laden Scaffold Printing 26111.2.2.2 Synchronous Bioprinting (Bioink and Sacrificial Ink Half and Half) 26111.2.3 Heterogeneous Independent/Interconnected Porous Structure Printing 26211.2.4 Long-term Perfusion Culture on a Chip 26511.2.5 Discussions (Properties, Pros, Cons, etc.) 26511.3 Large Scale Cell-laden Structures with Vascular Channel Printing 26611.3.1 Sacrificial Bioprinting 26611.3.2 Coaxial Bioprinting 26711.4 One-step Coaxial/Sacrificial Printing of Large Scale Vascularized Tissue Constructs 26811.4.1 Mechanism 26811.4.2 Freeform Structure with Vascular Channels Printing 26911.4.3 Heterogeneous Structure with Vascular Channels Printing 27011.4.4 Long-term Perfusion Culture on a Chip 27211.4.5 Discussion (Properties, Pros and Cons, etc.) 27211.5 Advanced Bioprinting Technique-Enabled Printing Highly Biomimetic Tissues 27311.5.1 Support Bath-Assisted Bioprinting 27311.5.2 Light-Based Bioprinting 27311.5.3 Discussion (Properties, Pros and Cons, etc.) 27511.6 Representative Biomedical Applications 275References 27612 3D Printing of Vascular Chips 28112.1 Introduction 28112.2 Construction Process of Hydrogel-Based Vascular Chips 28212.2.1 Damage-Free Demolding Process Based on Soft Fiber Template 28212.2.1.1 Damage-Free Demolding Process 28312.2.1.2 Comparative Analysis of Damage-Free and Conventional Demolding Processes 28312.2.2 Hydrogel Bonding Strategy Based on Twice-Cross-linking Mechanism 28612.2.2.1 Manufacturing Process of Hydrogel-Based Microfluidic Chips 28712.2.2.2 Mechanism Study 28712.2.2.3 Material Selection 28812.2.2.4 Feasible Domain 28912.2.2.5 Bonding Results 28912.2.3 Multi-Scale 3D Printing Process 29112.2.3.1 Mechanism of Multi-Scale 3D Printing Process 29112.2.3.2 Printing Parameters 29212.2.4 Construction Process of Hydrogel-Based Vascular Chips 29312.3 Characterization of Vascular Chips 29512.3.1 Fundamental Characterization of Vascular Chips 29512.3.1.1 Characterization of Endothelium Function of Channels 29512.3.1.2 Characterization of Endothelial Cells Viability 29512.3.1.3 Characterization of Endothelial Cells Morphology 29612.3.1.4 Characterization of Endothelium Channel 29712.3.2 Morphology Characterization of Hydrogel-Based Vascular Chips 29812.3.2.1 Multi-Level Bifurcated Channel Network Structure 29812.3.2.2 Multi-Scale Vascular Model 29912.3.2.3 Biomimicking Vascular Model 29912.3.3 Characterization of Vascular Function 30212.3.3.1 Nutrition Supply Function 30212.3.3.2 Expression of Key Functional Proteins in Endothelial Cells 30212.3.3.3 Simulation of Vascular Inflammation Reaction 30312.3.3.4 Characterization of Vascular Barrier Function 30412.4 Conclusion 307References 30813 3D Printing of In Vitro Models 31113.1 Introduction 31113.2 Typical 3D Bioprinting Technologies and Common Target Tissue/Organ Demand 31213.2.1 Inkjet-Based Bioprinting 31313.2.2 Extrusion-Based Bioprinting 31413.2.3 Light-Assisted Bioprinting 31513.3 Developing Process of In Vitro Models 31613.3.1 Mini-Tissue in 3D Growth State 31613.3.1.1 Sphere Mini-Tissue Model 31613.3.1.2 Fiber Mini-Tissue Model 31713.3.1.3 Array Mini-Tissue Model 31813.3.1.4 Limitations 31913.3.2 Organ-on-a-Chip with Multiplex Microenvironment 31913.3.2.1 Integrated Organ-on-a-Chip 32113.3.2.2 Modular Microfluidic System 32213.3.2.3 Multiple-Organ System 32313.3.2.4 Limitations 32513.3.3 Tissue/Organ Construct with Biomimicking Property 32513.3.3.1 Vascular Construct 32613.3.3.2 Vascularized Tissue Construct 32813.3.3.3 Limitations 33013.4 3D Printing of In Vitro Tumor Models 33013.4.1 Tumor Cell-Laden Construct 33013.4.2 Multi-Cell Tumor Sphere 33113.4.3 Tumor Metastasis Model with Angiogenesis 33213.5 Summary and Prospect 33413.5.1 Key Virtue and Comparison 33413.5.2 Outlook 33413.5.2.1 3D Bioprinting Technology 33513.5.2.2 Individual Differences 33513.5.2.3 Systematic Interaction 33513.5.2.4 Industrialization 33513.6 Conclusions 336References 33614 Protocol of Typical 3D Bioprinting 339Reference 343Index 345