Methods in Bioengineering: 3D Tissue Engineering Berthiaume, Francois ISBN-13: 9781596934580 Table of Contents Preface Chapter 1. Chemical Modification of Porous Scaffolds Using Plasma Polymers 1.1. Introduction 1.2. Experimental Design 1.3. Materials 1.4. Methods 1.4.1. Scaffold preparation 1.4.2. Deposition of plasma polymers 1.4.3. Surface analysis 1.4.4. Cell culture on scaffolds 1.4.5. Alamar Blue assay 1.4.6. Cell viability assay on scaffolds 1.4.7. Scanning electron microscopy 1.4.8. Microcomputed tomography of scaffolds 1.5. Data Acquisition, Anticipated Results, and Interpretation 1.5.1. Surface analysis 1.5.2. Investigation of cell culture on modified scaffolds 1.6. Discussion and Commentary 1.7. Application Notes 1.8. Summary Points Chapter 2. Three-Dimensional Cultures in Soft Self-Assembling Nanofibers 2.1. Introduction 2.2. Experimental Design 2.3. Materials 2.3.1. Reagents 2.3.2. Equipment 2.4. Methods 2.4.1. Self-assembling peptide preparation 2.4.2. Cell encapsulation into the self-assembling peptide 2.4.3. Sandwich method 2.4.4. Cell isolation and culture of isolated cells 2.4.5. Cryosections of the 3D cultures 2.4.6. Cell proliferation study using 5-bromodeoxyuridine (Brd U) uptake analysis 2.4.7. Cell viability 2.4.8. Protein analysis 2.4.9. Sample staining 2.4.10. sgag quantification 2.4.11. Lysis of 3D cultures for RNA extraction 2.5. Data Acquisition, Anticipated Results, and Interpretation 2.6. Discussion and Commentary 2.7. Troubleshooting 2.8. Application Notes 2.9. Summary Points Chapter 3. 3D Fibrin Matrices as Scaffold for Depot and Release of Bioactive Molecules 3.1. Introduction 3.2. Experimental Design 3.3. Materials 3.3.1. Chemicals 3.3.2. Equipment/Infrastructure 3.4. Methods 3.4.1. Preparation of 3D fibrin matrices 3.4.2. Introduction of bioactive molecules 3.4.3. Cell Culture
3.4.4. Data acquisition, important controls, and staining procedures 3.5. Data Analysis, Anticipated Results, and Interpretation 3.6. Discussion and Commentary 3.7. Application Notes 3.8. Summary Points Selected Bibliography Chapter 4. Designer Self-Assembling Peptide Scaffolds for 3D Tissue Cell Cultures 4.1. Introduction 4.1.1. Discovery and development of self-assembling peptide scaffolds 4.1.2. The nanofiber structure of the peptide scaffold 4.1.3. A generic biological scaffold 4.1.4. Peptide scaffold fosters chondrocyte extracellular matrix production 4.1.5. Designer peptides appended with active motifs 4.2. Materials 4.3. Reagents 4.4. Methods 4.4.1. Peptide solution preparation 4.4.2. Designer peptide synthesis and scaffold preparation 4.4.3. Culture cells in plate inserts 4.4.4. Cell culture system 4.4.5. Neural cell culture and seeding 4.4.6. Preparation of MC3T3-E1 cells 4.4.7. Cell culture of human umbilical vein endothelial cells (HUVECs) 4.4.8. Cell proliferation assay 4.4.9. DNA content measurement 4.4.10. Boundary-sandwiched cell migration assay 4.4.11. Fluorescence microscopy 4.4.12. Immunocytochemistry 4.4.13. SEM sample preparation 4.4.14. Circular dichroism (CD) 4.4.15. Structural study using atomic force microscopy (AFM) 4.4.16. Biomechanical study using rheology 4.4.17. Alkaline phosphatase (ALP) staining for MC3T3-E1 cells 4.4.18. Biochemical assays for alkaline phosphatase (ALP) activity for MC3T3-E1 cells 4.4.19. Low protein release from the peptide scaffold 4.5. Data Acquisition, Results, and Interpretation 4.5.1. Designer self-assembling peptide nanofiber hydrogel scaffold 4.5.2. 3D cell cultures 4.5.3. Cell migration in peptide scaffolds 4.5.4. Rheology of peptide hydrogel scaffold 4.5.5. Tissue regeneration and tissue engineering 4.5.6. Protein releases from the peptide nanofiber hydrogel scaffold 4.6. Discussions and Commentary 4.7. Application Notes 4.7.1. In vivo injectable self-assembling peptides 4.7.2. In vitro multicell system for tissue engineering 4.7.3. Mixed peptide hydrogel with polymer composites 4.8. Summary Points Chapter 5. Chip-Based Tissue Engineering in Microbioreactors 5.1. Introduction 5.2. Experimental Design 5.3. Materials and Equipment 5.3.1. Fabrication of p- and f-chips 5.3.2. Fabrication of the r-chip 5.3.3. Bioreactor assembly 5.4. Methods 5.4.1. KITChip fabrication (p- and f-chip) 5.4.2. r-chip (SMART technology) 5.4.3. Preparation of KITChips and bioreactors for cell culture 5.4.4. Cell culture 5.4.5. Data analysis 5.5. Anticipated Results 5.6. Discussion and Commentary 5.7. Application Notes
5.8. Summary Points Chapter 6. Affinity-Binding Alginate Scaffolds for the Controlled Delivery of Multiple Heparin-Binding Proteins 6.1. Introduction 6.2. Experimental Design 6.3. Materials 6.3.1. Materials for alginate sulfation 6.3.2. Materials for scaffold fabrication and factor loading 6.3.3. Materials for immunohistochemistry 6.4. Methods 6.4.1. General procedure 6.4.2. Preparation of alginate-sulfate and characterization of product 6.4.3. Scaffold fabrication 6.4.4. Triple factor loading into scaffolds and release studies 6.4.5. In vivo studies: immunostaining, imaging, and data acquisition 6.5. Anticipated Results 6.6. Discussion and Commentary 6.7. Application Notes 6.8. Summary Points Chapter 7. Self-Assembly of Cell-Laden Hydrogels on the Liquid-Air Interface 7.1. Introduction 7.2. Experimental Design 7.3. Materials 7.4. Methods 7.4.1. Preparation of 1m of 20% poly (ethylene glycol) diacrylate prepolymer 7.4.2. Cell preparation (NIH 3T3) 7.4.3. Storage of PEG 7.4.4. Mixing PEG solution with cells 7.4.5. Glass OTS treatment 7.4.6. Photolithography 7.4.7. Aggregation process 7.4.8. Data analysis 7.5. Anticipated Results 7.6. Discussion and Commentary 7.7. Application Notes 7.8. Summary Points Chapter 8. 3D Encapsulation of Cells in Hydrogels Using Radical and Addition Cross-Linking 8.1. Introduction 8.2. Experimental Design 8.3. Materials 8.4. Methods 8.4.1. Preparation of materials 8.4.2. Initiation of pendant addition reactions and preparation of cells for encapsulation 8.4.3. Synthesis of hydrogels 8.4.4. Characterization of hydrogels 8.4.5. Characterization of cellular behavior in gels 8.5. Anticipated Results 8.6. Discussion and Commentary 8.7. Application Notes 8.8. Summary Points Chapter 9. Micromolded Nonadhesive Hydrogels for the Self-Assembly of Scaffold-Free 3D Cellular Microtissues 9.1. Introduction 9.2. Experimental Design 9.3. Materials 9.4. Methods 9.4.1. Design and fabrication of micromolds 9.4.2. Casting of agarose micromolded hydrogels 9.4.3. Casting polyacrylamide micromolded hydrogels 9.4.4. Formation of 3D microtissues 9.4.5. Side-on viewing of self-assembly 9.4.6. Spheroid size quantification 9.4.7. Live cell fluorescent staining and determination of self-sorting patterns of cells
9.4.8. WST-1 cell proliferation assay adapted for microtissues 9.4.9. Harvesting microtissues 9.4.10. LIVE/DEAD staining to determine microtissue viability 9.4.11. Data analysis 9.5. Anticipated Results 9.6. Discussion and Commentary 9.7. Application Notes 9.8. Summary Points Chapter 10. On-Demand 3D Freeform Fabrication of Tissue Structures Using Bioprinting 10.1. Introduction 10.2. Experimental Design 10.3. Materials 10.3.1. Reagents 10.3.2. Facilities/equipment 10.3.3. Overview of the robotic 3D bioprinter 10.3.4. Software for the 3D bioprinter operations 10.3.5. Sterilization of the fluidic pathways in the 3D bioprinter for biomaterial printing 10.3.6. Loading of materials to the printer 10.3.7. Control of droplet dispensing 10.3.8. Cleaning of the printing path and microvalve after use 10.4. Methods 10.4.1. Optimization of the printing resolution for a cell-laden single-layer hydrogel scaffold 10.4.2. Multilayer cell-laden hydrogel scaffold 10.4.3. Cell-laden hydrogel scaffold on a nonplanar surface 10.4.4. Staining and imaging 10.5. Anticipated Results, Data Acquisition, and Interpretation 10.6. Discussion and Commentary 10.7. Application Notes 10.8. Summary Points Chapter 11. Three-Dimensional Neuronal Cultures 11.1. Introduction 11.1.1. 2D versus 3D culture models 11.1.2. Cell type and culture configurations 11.2. Experimental Design 11.3. Materials 11.3.1. Harvest 11.3.2. Dissociation 11.3.3. Plating 11.3.4. Assessment 11.4. Methods 11.4.1. Harvest 11.4.2. Dissociation 11.4.3. Plating 11.4.4. General assessment: staining, imaging, and data acquisition 11.4.5. Data analysis 11.5. Anticipated Results and Discussion 11.5.1. Characterization of cell morphology and viability in 3D neuronal cultures 11.5.2. Cell considerations 11.5.3. Scaffold considerations 11.6. Application Notes and Commentary 11.7. Summary Points Chapter 12. Engineering Cartilage Tissue with Zonal Properties 12.1. Introduction 12.2. Materials 12.2.1. Reagents/supplies 12.2.2. Facilities/equipment 12.3. Methods 12.3.1. Obtaining and tracking zonal chondrocytes 12.3.2. Zonal construct formation 12.3.3. Analyses 12.4. Discussion and Commentary 12.5. Summary Points
Chapter 13. Cartilage and Synovial Joint Regeneration by Cell Homing in Bioprinted, Anatomically Correct 3D Scaffolds 13.1. Introduction 13.2. Experimental Design 13.3. Materials 13.4. Methods 13.4.1. Bioplotting PCL-HA scaffolds for synovial joint condyle tissue engineering 13.4.2. Infusing TGFβ3 in a collagen gel into the microchannels of a PCL-HA scaffold 13.5. Results and Interpretation 13.6. Discussion and Commentary 13.7. Application Notes 13.8. Summary Points Chapter 14. Integration of Experimental and Computational Microfluidics in 3D Tissue Engineering 14.1. Introduction 14.2. Experimental Design 14.3. Materials 14.3.1. Solutions 14.3.2. Disposables 14.3.3. Equipment 14.3.4. Custom equipment 14.3.5. Software 14.4. Methods 14.4.1. Microfluidic chamber design 14.4.2. Computational model 14.4.3. Dynamic scaffold seeding 14.4.4. Microbioreactor setup 14.4.5. Imaging and data acquisition 14.5. Anticipated Results and Interpretation 14.6. Discussion and Commentary 14.7. Application Notes 14.8. Summary Points About the Editors Index