Tissue Engineering: A Primer with Laboratory Demonstrations

274 Pages·2021·17.994 MB·English

by  Jeong-Yeol Yoon

Jeong-Yeol Yoon
Tissue
Engineering
A Primer with Laboratory
Demonstrations
Tissue Engineering
Jeong-Yeol Yoon
Tissue Engineering
A Primer with Laboratory Demonstrations
Jeong-Yeol Yoon
Department of Biomedical Engineering
The University of Arizona
Tucson, AZ, USA
ISBN 978-3-030-83695-5 ISBN 978-3-030-83696-2 (eBook)
https://doi.org/10.1007/978-3-030-83696-2
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Preface
I have been teaching a semester-long upper-division undergraduate college class on
tissue engineering. There are several good textbooks, albeit not many, on tissue
engineering. (This is understandable since tissue engineering is still a new disci-
pline.) However, many textbooks are exceptionally long, close to a thousand pages
or even two thousand pages. They are written by many authors, in a manner of an
edited book, potentially lacking the following: consistency, explanations of basic
terms, example questions, and engineering design/calculation.
In addition, I have been a firm believer that the core bioengineering and related-
discipline classes are to be delivered together with the hands-on laboratory exer-
cises. I have already demonstrated this concept in my other textbook, Introduction
to Biosensors, whose second edition was also published by Springer in 2016.
With this book, I offer a single-authored primer textbook on tissue engineering,
not in the manner of a lengthy edited book. It covers the fundamental basics of tis-
sue engineering in a concise manner, accompanied by a series of laboratory exer-
cises. While we can certainly use this book for a class with laboratory exercises, we
can also use it without laboratories. A list of questions and discussion topics are
added to each laboratory exercise. The instructor can lead such discussion collab-
oratively using the experimental procedures and results included in the textbook. As
the laboratory results are included with actual images and data for all laboratory
exercises, scientists and engineers not in colleges can also learn the concepts in a
hands-on and visual manner to better understand concepts. It will also lay a founda-
tion to build their experiments towards their research and commercial development.
With this book, I aimed to accomplish the followings:
– Most up-to-date aspects of tissue engineering are covered in a concise manner
while providing easy-to-understand basic concepts.
– Step-by-step learning of all necessary concepts is provided.
– A large number of figures are provided. There are 226 figures through 14 chap-
ters in this book.
– Simple, low-cost, and easy-to-implement laboratory exercises are included in all
chapters, except for the first and last chapters.
v
vi Preface
– Photographs of all equipment and results of all laboratories are provided to visu-
alize tissue engineering concepts. These can be used as guides for practical labo-
ratory exercises towards better understanding the concepts or starting points
towards further research and development.
I sincerely thank my graduate teaching assistants at the University of Arizona.
They have helped me create and implement the laboratory exercises in my Cell and
Tissue Engineering class in the spring semesters from 2015 to 2021. I specifically
appreciate the following graduate teaching assistants: Dr. Katherine Klug (currently
at Davids Engineering), Dr. Soohee Cho (currently at Abbott), Dr. Tiffany-Heather
Ulep (currently at Roche), and Dr. Kattika Kaarj (presently a faculty member at
Mahidol University in Thailand), who had laid foundations in these laboratory exer-
cises. The following graduate teaching assistants have also helped establish the
laboratory exercises: Dr. Soo Chung (presently at United States Department of
Agriculture – Agricultural Research Service), Mr. Kenneth Schackart (currently at
the University of Arizona), and Mr. Ryan Zenhausern (currently at Georgia Institute
of Technology). Other graduate teaching assistants, Mr. Christopher Camp and Ms.
Carissa Grijalva, both at the University of Arizona, have also contributed substan-
tially to the laboratory exercises. I also thank all students who took my cell and
tissue engineering class for providing critical feedback and corrections.
I also thank my former and current department heads, Dr. Urs Utzinger and Dr.
Arthur Gmitro in the Biomedical Engineering Department. They have supported my
class by providing personnel, equipment, and laboratory space at the University of
Arizona. Support and suggestions from the editorial office at Springer, especially
Michael McCabe, are greatly appreciated. Finally, I want to sincerely thank my
wife, Dr. Sunhi Choi (Mathematics Department at the University of Arizona), for
her continuous inspiration and support during the preparation of this textbook.
Tucson, AZ, USA Jeong-Yeol Yoon
Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
1.1 Narrow Definition of Tissue Engineering . . . . . . . . . . . . . . . . . . . . 1
1.2 Early Attempt in Scaffold Development: Decellularized Matrix . . . 2
1.3 Simple TE Transplant Example: Skin . . . . . . . . . . . . . . . . . . . . . . . 4
1.4 Simple TE Transplant Example: Pancreas . . . . . . . . . . . . . . . . . . . . 6
1.5 Expanded Definition of Tissue Engineering: Organ-on- a-Chip
(OOC) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
1.6 Overview of This Book . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
2 Cell Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
2.1 What Is Cell Culture? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
2.2 Cell Physiology: Cell Membrane and Cytoskeleton . . . . . . . . . . . . 15
2.3 Focal Adhesion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
2.4 Cell Classification: Anchorage-Dependent Versus Anchorage-
Independent Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
2.5 Cell Classification: Normal Versus Immortalized Cells. . . . . . . . . . 19
2.6 Cell Classification: Normal Versus Stem Cells . . . . . . . . . . . . . . . . 22
2.7 Maintaining Sterile Environment: Biosafety Cabinet . . . . . . . . . . . 22
2.8 Maintaining Sterile Environment: Autoclave . . . . . . . . . . . . . . . . . . 23
2.9 Cell Culture: CO Incubator . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
2
2.10 Cell Imaging: Fluorescence Microscope . . . . . . . . . . . . . . . . . . . . . 25
2.11 Laboratory Task 1: Bacterial Cell Culture . . . . . . . . . . . . . . . . . . . . 26
2.12 Laboratory Task 2: Fluorescence Microscopic Imaging
of Mammalian Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
Reference . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
3 Cell Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
3.1 What Is Cell Metabolism? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
3.2 Energy Currency: ATP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
3.3 Cell Metabolism: Glycolysis and TCA Cycle . . . . . . . . . . . . . . . . . 35
3.4 Glucose Transport and Uptake . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
vii
viii Contents
3.5 Cell Metabolism: Glutaminolysis and TCA Cycle . . . . . . . . . . . . . 39
3.6 Glutamine Transport and Uptake . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
3.7 Role of Oxygen in Cell Metabolism . . . . . . . . . . . . . . . . . . . . . . . . 40
3.8 Culture Media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42
3.9 Cell Feeding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
3.10 Cell Passaging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
3.11 Cell Growth Kinetics: First-Order Growth Model . . . . . . . . . . . . . . 45
3.12 Laboratory Task 1: Media Preparation and Cell Feeding . . . . . . . . 47
3.13 Laboratory Task 2: Cell Passaging . . . . . . . . . . . . . . . . . . . . . . . . . . 51
Reference . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
4 Cell Imaging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
4.1 Overview of Fluorescence Microscopy . . . . . . . . . . . . . . . . . . . . . . 55
4.2 Fluorescence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56
4.3 Fluorescent Dyes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
4.4 Bioreceptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62
4.5 Fluorescence Microscope . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
4.6 Photobleaching . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67
4.7 Smartphone Fluorescence Microscope . . . . . . . . . . . . . . . . . . . . . . 68
4.8 Laboratory Task 1: Fluorescence Imaging of Nucleus and
Cytoskeleton . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70
4.9 Laboratory Task 2 (Alternative): Fluorescence Imaging
of Nucleus, Cytoskeleton, and Focal Adhesion . . . . . . . . . . . . . . . . 78
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
5 Stem Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81
5.1 What Are Stem Cells? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81
5.2 Why Do We Need Stem Cells for Tissue Engineering? . . . . . . . . . . 83
5.3 Embryonic Versus Adult Stem Cells . . . . . . . . . . . . . . . . . . . . . . . . 83
5.4 Use of Embryonic Stem Cells for Tissue Engineering Applications 85
5.5 Induced Pluripotent Stem Cells (iPSCs) . . . . . . . . . . . . . . . . . . . . . 88
5.6 Isolation of Stem Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89
5.7 Culturing Stem Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90
5.8 Morphogenetic Factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92
5.9 Hazards of Stem Cell Differentiation . . . . . . . . . . . . . . . . . . . . . . . . 93
5.10 Laboratory Task 1: Culturing Stem Cells . . . . . . . . . . . . . . . . . . . . 94
5.11 Laboratory Task 2: Embryoid Body Formation . . . . . . . . . . . . . . . . 95
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97
6 Biomaterial Surfaces . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99
6.1 Development of Biomaterial Surfaces for Tissue Engineering . . . . 99
6.2 Size and Shape Requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100
6.3 Synthetic Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102
6.4 Natural Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103
6.5 Nonspecific Cell–Surface Interactions . . . . . . . . . . . . . . . . . . . . . . . 106
6.6 Specific Cell–Surface Interactions . . . . . . . . . . . . . . . . . . . . . . . . . . 107
Contents ix
6.7 Bone Biomaterials, Apatite, and Bioglass . . . . . . . . . . . . . . . . . . . . 107
6.8 Hydrogels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110
6.9 Biodegradable Scaffolds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110
6.10 Encapsulation Scaffolds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112
6.11 Laboratory Task 1: Preparation of Various Biomaterial Surfaces . . 112
6.12 Laboratory Task 2: Contact Angle Measurements . . . . . . . . . . . . . . 117
6.13 Laboratory Task 3: Surface Roughness Measurements . . . . . . . . . . 119
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121
7 Focal Adhesion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123
7.1 What Is Focal Adhesion? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123
7.2 Cell Adhesion and Proliferation on Biomaterial Surfaces . . . . . . . . 125
7.3 Cell Migration After Focal Adhesion . . . . . . . . . . . . . . . . . . . . . . . . 127
7.4 Morphogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130
7.5 Laboratory Task 1: Fluorescence Staining of Focal Adhesion . . . . . 131
7.6 Laboratory Task 2: Fluorescence Imaging of Focal Adhesion . . . . . 132
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135
8 Contact Guidance and Cell Patterning . . . . . . . . . . . . . . . . . . . . . . . . . 137
8.1 What Is Contact Guidance? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137
8.2 Contact Guidance on Basement Membrane . . . . . . . . . . . . . . . . . . . 138
8.3 Nanogrooves . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139
8.4 Shear Flow-Resistant Composite Nanosurfaces . . . . . . . . . . . . . . . 142
8.5 Contact Guidance by Flow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144
8.6 Cell Patterning via Lithographic Protein Patterns . . . . . . . . . . . . . . 144
8.7 Cell Patterning via Direct Protein Deposition . . . . . . . . . . . . . . . . . 145
8.8 Cell Patterning via Microcontact Printing . . . . . . . . . . . . . . . . . . . . 146
8.9 Direct Cell Patterning via Inkjet Printing (Cell Printing) . . . . . . . . 147
8.10 Laboratory Task 1: Contact Guidance on Microgroove . . . . . . . . . . 149
8.11 Laboratory Task 2: Cell Patterning via Droplet Collagen
Deposition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 154
9 3D Scaffold Fabrication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155
9.1 3D Scaffolds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155
9.2 Electrospinning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 156
9.3 Materials Selection for Electrospinning Toward Tissue Engineering 158
9.4 Modification of Electrospun Fibers for Tissue Engineering . . . . . . 159
9.5 Contact Guidance on Electrospun Fibers . . . . . . . . . . . . . . . . . . . . . 159
9.6 Controlled Release of Growth Factors and Bioactive Factors from
Electrospun Fibers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161
9.7 Core–Shell Structured Electrospun Fibers . . . . . . . . . . . . . . . . . . . . 161
9.8 Addition of GAG-Like Structure to Electrospun Fibers . . . . . . . . . 162
9.9 3D Printed Scaffolds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163
9.10 Various 3D Printing Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 164
9.11 Hydrogel Bioprinting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167
x Contents
9.12 Adding Porosity and Nanostructure to 3D Printing . . . . . . . . . . . . . 168
9.13 Indirect 3D Printing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169
9.14 Laboratory Task 1: Electrospinning . . . . . . . . . . . . . . . . . . . . . . . . . 169
9.15 Laboratory Task 2: 3D Printing . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173
10 Design of In Vitro Culture and Bioreactor . . . . . . . . . . . . . . . . . . . . . . 175
10.1 Design of In Vitro Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175
10.2 Doubling Time (td) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 176
10.3 Mean Residence Time (t ) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 179
res
10.4 Oxygen Depletion Time (t ) . . . . . . . . . . . . . . . . . . . . . . . . . . . 181
O2dep
10.5 Oxygen Diffusion Time (t ) . . . . . . . . . . . . . . . . . . . . . . . . . . . 183
O2diff
10.6 Design of In Vitro Culture Considering Both Oxygen
Depletion Time and Oxygen Diffusion Time . . . . . . . . . . . . . . . . . 185
10.7 Design of Tissue-Engineered Device Using Characteristic Time . 187
10.8 Tissue Engineering Bioreactor . . . . . . . . . . . . . . . . . . . . . . . . . . . . 187
10.9 Measurements from a Tissue Engineering Bioreactor . . . . . . . . . . 190
10.10 Laboratory Task 1: Design of In Vitro Culture and Co-culture . . . 190
10.11 Laboratory Task 2: Design of Artificial Vasculature Using
Oxygen Depletion and Diffusion Times . . . . . . . . . . . . . . . . . . . . 191
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 192
11 Organ-on-a-Chip . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 193
11.1 2D Versus 3D Cell Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 193
11.2 Organ-on-a-Chip . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 194
11.3 How Do You Fabricate Lab-on-a-Chip (LOC)? . . . . . . . . . . . . . . . 195
11.4 OOC Example: Kidney-on-a-Chip . . . . . . . . . . . . . . . . . . . . . . . . 198
11.5 OOC Example: Liver-on-a-Chip . . . . . . . . . . . . . . . . . . . . . . . . . . 200
11.6 OOC Example: Lung-on-a-Chip . . . . . . . . . . . . . . . . . . . . . . . . . . 203
11.7 OOC Example: Angiogenesis-on-a-Chip . . . . . . . . . . . . . . . . . . . . 204
11.8 OOC Example: Blood–Brain Barrier (BBB)-on-a-Chip . . . . . . . . 205
11.9 Other OOC Examples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 206
11.10 Multiple-Organs-on-a-Chip and Human-on-a-Chip . . . . . . . . . . . 206
11.11 OOC Application: Drug Testing . . . . . . . . . . . . . . . . . . . . . . . . . . 207
11.12 OOC Application: Disease Model . . . . . . . . . . . . . . . . . . . . . . . . . 208
11.13 Mechanical Stimuli to OOCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 209
11.14 Laboratory Task 1: OOC Fabrication . . . . . . . . . . . . . . . . . . . . . . . 210
11.15 Laboratory Task 2: Drug Testing with OOC . . . . . . . . . . . . . . . . . 213
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 216
12 Tissue-Engineered Skin Transplant . . . . . . . . . . . . . . . . . . . . . . . . . . . 219
12.1 When Do You Need Skin Transplants? . . . . . . . . . . . . . . . . . . . . . 219
12.2 Basic Anatomy of Skin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 221
12.3 How Can We Culture Keratinocytes In Vitro? . . . . . . . . . . . . . . . . 222
12.4 Langerhans Cells and Immune Response of Skin . . . . . . . . . . . . . 222