Methods in Bioengineering: 3D Tissue Engineering

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1 Methods in Bioengineering: 3D Tissue Engineering Berthiaume, Francois ISBN-13: 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 Scaffold preparation Deposition of plasma polymers Surface analysis Cell culture on scaffolds Alamar Blue assay Cell viability assay on scaffolds Scanning electron microscopy Microcomputed tomography of scaffolds 1.5. Data Acquisition, Anticipated Results, and Interpretation Surface analysis 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 Reagents Equipment 2.4. Methods Self-assembling peptide preparation Cell encapsulation into the self-assembling peptide Sandwich method Cell isolation and culture of isolated cells Cryosections of the 3D cultures Cell proliferation study using 5-bromodeoxyuridine (Brd U) uptake analysis Cell viability Protein analysis Sample staining sgag quantification 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 Chemicals Equipment/Infrastructure 3.4. Methods Preparation of 3D fibrin matrices Introduction of bioactive molecules Cell Culture

2 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 Discovery and development of self-assembling peptide scaffolds The nanofiber structure of the peptide scaffold A generic biological scaffold Peptide scaffold fosters chondrocyte extracellular matrix production Designer peptides appended with active motifs 4.2. Materials 4.3. Reagents 4.4. Methods Peptide solution preparation Designer peptide synthesis and scaffold preparation Culture cells in plate inserts Cell culture system Neural cell culture and seeding Preparation of MC3T3-E1 cells Cell culture of human umbilical vein endothelial cells (HUVECs) Cell proliferation assay DNA content measurement Boundary-sandwiched cell migration assay Fluorescence microscopy Immunocytochemistry SEM sample preparation Circular dichroism (CD) Structural study using atomic force microscopy (AFM) Biomechanical study using rheology Alkaline phosphatase (ALP) staining for MC3T3-E1 cells Biochemical assays for alkaline phosphatase (ALP) activity for MC3T3-E1 cells Low protein release from the peptide scaffold 4.5. Data Acquisition, Results, and Interpretation Designer self-assembling peptide nanofiber hydrogel scaffold D cell cultures Cell migration in peptide scaffolds Rheology of peptide hydrogel scaffold Tissue regeneration and tissue engineering Protein releases from the peptide nanofiber hydrogel scaffold 4.6. Discussions and Commentary 4.7. Application Notes In vivo injectable self-assembling peptides In vitro multicell system for tissue engineering 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 Fabrication of p- and f-chips Fabrication of the r-chip Bioreactor assembly 5.4. Methods KITChip fabrication (p- and f-chip) r-chip (SMART technology) Preparation of KITChips and bioreactors for cell culture Cell culture Data analysis 5.5. Anticipated Results 5.6. Discussion and Commentary 5.7. Application Notes

3 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 Materials for alginate sulfation Materials for scaffold fabrication and factor loading Materials for immunohistochemistry 6.4. Methods General procedure Preparation of alginate-sulfate and characterization of product Scaffold fabrication Triple factor loading into scaffolds and release studies 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 Preparation of 1m of 20% poly (ethylene glycol) diacrylate prepolymer Cell preparation (NIH 3T3) Storage of PEG Mixing PEG solution with cells Glass OTS treatment Photolithography Aggregation process 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 Preparation of materials Initiation of pendant addition reactions and preparation of cells for encapsulation Synthesis of hydrogels Characterization of hydrogels 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 Design and fabrication of micromolds Casting of agarose micromolded hydrogels Casting polyacrylamide micromolded hydrogels Formation of 3D microtissues Side-on viewing of self-assembly Spheroid size quantification Live cell fluorescent staining and determination of self-sorting patterns of cells

4 WST-1 cell proliferation assay adapted for microtissues Harvesting microtissues LIVE/DEAD staining to determine microtissue viability 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 Introduction Experimental Design Materials Reagents Facilities/equipment Overview of the robotic 3D bioprinter Software for the 3D bioprinter operations Sterilization of the fluidic pathways in the 3D bioprinter for biomaterial printing Loading of materials to the printer Control of droplet dispensing Cleaning of the printing path and microvalve after use Methods Optimization of the printing resolution for a cell-laden single-layer hydrogel scaffold Multilayer cell-laden hydrogel scaffold Cell-laden hydrogel scaffold on a nonplanar surface Staining and imaging Anticipated Results, Data Acquisition, and Interpretation Discussion and Commentary Application Notes Summary Points Chapter 11. Three-Dimensional Neuronal Cultures Introduction D versus 3D culture models Cell type and culture configurations Experimental Design Materials Harvest Dissociation Plating Assessment Methods Harvest Dissociation Plating General assessment: staining, imaging, and data acquisition Data analysis Anticipated Results and Discussion Characterization of cell morphology and viability in 3D neuronal cultures Cell considerations Scaffold considerations Application Notes and Commentary Summary Points Chapter 12. Engineering Cartilage Tissue with Zonal Properties Introduction Materials Reagents/supplies Facilities/equipment Methods Obtaining and tracking zonal chondrocytes Zonal construct formation Analyses Discussion and Commentary Summary Points

5 Chapter 13. Cartilage and Synovial Joint Regeneration by Cell Homing in Bioprinted, Anatomically Correct 3D Scaffolds Introduction Experimental Design Materials Methods Bioplotting PCL-HA scaffolds for synovial joint condyle tissue engineering Infusing TGFβ3 in a collagen gel into the microchannels of a PCL-HA scaffold Results and Interpretation Discussion and Commentary Application Notes Summary Points Chapter 14. Integration of Experimental and Computational Microfluidics in 3D Tissue Engineering Introduction Experimental Design Materials Solutions Disposables Equipment Custom equipment Software Methods Microfluidic chamber design Computational model Dynamic scaffold seeding Microbioreactor setup Imaging and data acquisition Anticipated Results and Interpretation Discussion and Commentary Application Notes Summary Points About the Editors Index