Methods in Bioengineering: 3d Tissue Engineering (The Artech House Methods in Bioengineering)

279 Pages·2010·23.73 MB·English

by  Francois Berthiaume

Methods in Bioengineering
3D Tissue Engineering
The Artech House Methods in Bioengineering Series
Series Editors
Martin L.Yarmush, M.D., Ph.D.
RobertLanger, Sc.D.
Methods in Bioengineering: 3D Tissue Engineering,
FrançoisBerthiaumeand Jeffrey Morgan, editors
Methods in Bioengineering: Biomicrofabrication andBiomicrofluidics,
Jeffrey D.Zahnand Luke P. Lee, editors
Methods in Bioengineering: Microdevices in Biology and Medicine,
YaakovNahmias andSangeeta N.Bhatia, editors
Methods in Bioengineering: Nanoscale Bioengineering and Nanomedicine,
KaushalRegeand IgorMedintz, editors
Methods in Bioengineering: Stem Cell Bioengineering,
BijuParekkadanand Martin L. Yarmush, editors
Methods in Bioengineering: Systems Analysis of Biological Networks,
ArulJayaraman andJuergenHahn, editors
Translational Approaches in Tissue Engineering and Regenerative Medi-
cine, Jeremy Mao, GordanaVunjak-Novakovic,AntoniosG.Mikos, and
AnthonyAtala, editors
Methods in Bioengineering
3D Tissue Engineering
FrançoisBerthiaume
Department of Biomedical Engineering, Rutgers University
Jeffrey Morgan
Department of Molecular Pharmacology, Physiology & Biotechnology, Brown University
Editors
artechhouse.com
Library of Congress Cataloging-in-Publication Data
A catalog record for this book is available from the U.S. Library of Congress.
British Library Cataloguing in Publication Data
A catalogue record for this book is available from the British Library.
ISBN-13: 978-1-59693-458-0
Cover design by Vicki Kane
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Contents
Preface xv
CHAPTER 1
Chemical Modification of Porous Scaffolds Using Plasma Polymers 1
1.1 Introduction 2
1.2 Experimental Design 3
1.3 Materials 3
1.4 Methods 4
1.4.1 Scaffold preparation 4
1.4.2 Deposition of plasma polymers 5
1.4.3 Surface analysis 6
1.4.4 Cell culture on scaffolds 7
1.4.5 AlamarBlue assay 7
1.4.6 Cell viability assay on scaffolds 8
1.4.7 Scanning electron microscopy 8
1.4.8 Microcomputedtomography of scaffolds 9
1.5 Data Acquisition, Anticipated Results, and Interpretation 9
1.5.1 Surface analysis 9
1.5.2 Investigation of cell culture on modified scaffolds 11
1.6 Discussion and Commentary 14
1.7 Application Notes 17
1.8 Summary Points 18
Acknowledgments 18
References 18
CHAPTER 2
Three-Dimensional Cultures in Soft Self-AssemblingNanofibers 21
2.1 Introduction 22
2.2 Experimental Design 24
2.3 Materials 25
2.3.1 Reagents 25
2.3.2 Equipment 26
2.4 Methods 26
2.4.1 Self-assembling peptide preparation 26
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Contents
2.4.2 Cell encapsulation into the self-assembling peptide 26
2.4.3 Sandwich method 27
2.4.4 Cell isolation and culture of isolated cells 28
2.4.5 Cryosectionsof the 3D cultures 28
2.4.6 Cell proliferation study using5-bromodeoxyuridine(BrdU)
uptake analysis 29
2.4.7 Cell viability 29
2.4.8 Protein analysis 29
2.4.9 Sample staining 30
2.4.10 sGAGquantification 32
2.4.11 Lysis of 3D cultures for RNA extraction 33
2.5 Data Acquisition, Anticipated Results, and Interpretation 33
2.6 Discussion and Commentary 37
2.7 Troubleshooting 39
2.8 Application Notes 39
2.9 Summary Points 40
Acknowledgments 41
References 41
CHAPTER 3
3D Fibrin Matrices as Scaffold for Depot and Release ofBioactiveMolecules 43
3.1 Introduction 44
3.2 Experimental Design 45
3.3 Materials 46
3.3.1 Chemicals 46
3.3.2 Equipment/Infrastructure 46
3.4 Methods 46
3.4.1 Preparation of 3D fibrin matrices 46
3.4.2 Introduction ofbioactivemolecules 49
3.4.3 Cell Culture 50
3.4.4 Data acquisition, important controls, and staining procedures 51
3.5 Data Analysis, Anticipated Results, and Interpretation 53
3.6 Discussion and Commentary 54
3.7 Application Notes 55
3.8 Summary Points 55
Acknowledgments 56
References 56
Selected Bibliography 58
CHAPTER 4
Designer Self-Assembling Peptide Scaffolds for 3D Tissue Cell Cultures 59
4.1 Introduction 60
vi
Contents
4.1.1 Discovery and development of self-assembling
peptide scaffolds 61
4.1.2 Thenanofiberstructure of the peptide scaffold 61
4.1.3 A generic biological scaffold 62
4.1.4 Peptide scaffold fosterschondrocyteextracellular
matrix production 63
4.1.5 Designer peptides appended with active motifs 64
4.2 Materials 65
4.3 Reagents 66
4.4 Methods 66
4.4.1 Peptide solution preparation 66
4.4.2 Designer peptide synthesis and scaffold preparation 66
4.4.3 Culture cells in plate inserts 67
4.4.4 Cell culture system 67
4.4.5 Neural cell culture and seeding 67
4.4.6 Preparation of MC3T3-E1 cells 68
4.4.7 Cell culture of human umbilical vein endothelial cells (HUVECs) 68
4.4.8 Cell proliferation assay 68
4.4.9 DNA content measurement 69
4.4.10 Boundary-sandwiched cell migration assay 69
4.4.11 Fluorescence microscopy 70
4.4.12 Immunocytochemistry 70
4.4.13 SEM sample preparation 70
4.4.14 Circular dichroism (CD) 70
4.4.15 Structural study using atomic force microscopy (AFM) 71
4.4.16 Biomechanicalstudy using rheology 71
4.4.17 Alkalinephosphatase(ALP) staining for MC3T3-E1 cells 71
4.4.18 Biochemical assays for alkalinephosphatase(ALP)
activity for MC3T3-E1 cells 71
4.4.19 Low protein release from the peptide scaffold 72
4.5 Data Acquisition, Results, and Interpretation 72
4.5.1 Designer self-assembling peptidenanofiberhydrogelscaffold 72
4.5.2 3D cell cultures 73
4.5.3 Cell migration in peptide scaffolds 74
4.5.4 Rheology of peptidehydrogelscaffold 75
4.5.5 Tissue regeneration and tissue engineering 76
4.5.6 Protein releases from the peptidenanofiberhydrogelscaffold 76
4.6 Discussions and Commentary 78
4.7 Application Notes 79
4.7.1 In vivo injectable self-assembling peptides 79
4.7.2 In vitromulticellsystem for tissue engineering 80
4.7.3 Mixed peptidehydrogelwith polymer composites 80
4.8 Summary Points 80
Acknowledgments 81
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Contents
References 81
CHAPTER 5
Chip-Based Tissue Engineering inMicrobioreactors 83
5.1 Introduction 84
5.2 Experimental Design 86
5.3 Materials and Equipment 86
5.3.1 Fabrication of p- and f-chips 86
5.3.2 Fabrication of the r-chip 86
5.3.3 Bioreactorassembly 87
5.4 Methods 91
5.4.1 KITChipfabrication (p- and f-chip) 91
5.4.2 r-chip (SMART technology) 92
5.4.3 Preparation ofKITChipsandbioreactorsfor cell culture 93
5.4.4 Cell culture 94
5.4.5 Data analysis 95
5.5 Anticipated Results 95
5.6 Discussion and Commentary 96
5.7 Application Notes 97
5.8 Summary Points 98
Acknowledgments 98
References 99
CHAPTER 6
Affinity-BindingAlginateScaffolds for the Controlled Delivery of
Multiple Heparin-Binding Proteins 101
6.1 Introduction 102
6.2 Experimental Design 104
6.3 Materials 105
6.3.1 Materials foralginatesulfation 105
6.3.2 Materials for scaffold fabrication and factor loading 105
6.3.3 Materials forimmunohistochemistry 105
6.4 Methods 106
6.4.1 General procedure 106
6.4.2 Preparation of alginate-sulfate and characterization
of product 106
6.4.3 Scaffold fabrication 109
6.4.4 Triple factor loading into scaffolds and release studies 110
6.4.5 In vivo studies:immunostaining, imaging, and data acquisition 110
6.5 Anticipated Results 112
6.6 Discussion and Commentary 112
6.7 Application Notes 117
6.8 Summary Points 117
viii
Contents
Acknowledgments 118
References 118
CHAPTER 7
Self-Assembly of Cell-LadenHydrogelson the Liquid-Air Interface 121
7.1 Introduction 122
7.2 Experimental Design 123
7.3 Materials 124
7.4 Methods 124
7.4.1 Preparation of 1m of 20% poly (ethylene glycol)
diacrylateprepolymer 124
7.4.2 Cell preparation (NIH 3T3) 124
7.4.3 Storage of PEG 125
7.4.4 Mixing PEG solution with cells 125
7.4.5 Glass OTS treatment 125
7.4.6 Photolithography 125
7.4.7 Aggregation process 126
7.4.8 Data analysis 127
7.5 Anticipated Results 127
7.6 Discussion and Commentary 127
7.7 Application Notes 129
7.8 Summary Points 129
Acknowledgments 130
References 130
CHAPTER 8
3D Encapsulation of Cells inHydrogelsUsing Radical and Addition Cross-Linking 133
8.1 Introduction 134
8.2 Experimental Design 136
8.3 Materials 136
8.4 Methods 137
8.4.1 Preparation of materials 137
8.4.2 Initiation of pendant addition reactions and preparation of
cells for encapsulation 138
8.4.3 Synthesis ofhydrogels 139
8.4.4 Characterization ofhydrogels 140
8.4.5 Characterization of cellular behavior in gels 141
8.5 Anticipated Results 145
8.6 Discussion and Commentary 146
8.7 Application Notes 146
8.8 Summary Points 147
References 147
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