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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
© Springer Nature Switzerland AG 2022

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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: CO2 Incubator�� 24
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
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
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
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 (tres) �� 179
10.4 Oxygen Depletion Time (tO2dep)�� 181
10.5 Oxygen Diffusion Time (tO2diff) �� 183
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
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
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
Contents xi
12.5 Scaffold for Tissue-Engineered Skin Transplant�� 223

12.6 Use of Stem Cells for TE Skin Transplant�� 226
12.7 Laboratory Task 1: Construction of Skin TE Scaffold�� 227
12.8 Laboratory Task 2: Seeding and Proliferating Keratinocytes
on the TE Skin Scaffold�� 227
12.9 Laboratory Task 3: Force: Deflection Curve of the TE Skin
Transplant�� 228
13 Vascularization of Tissue Transplants�� 235
13.1 Angiogenesis and Vascularization�� 235
13.2 Anatomy and Physiology of Blood Vessel�� 236
13.3 Process of Angiogenesis �� 238
13.4 Angiogenesis-Stimulating Growth Factors and Angiogenesis
Inhibitors �� 239
13.5 HUVEC�� 241
13.6 Formation of Vasculature Network �� 241
13.7 Alignment of Vascular Endothelial Cells to Flow�� 243
13.8 Laboratory Task 1: Vessel Sprouting by VEGF on Paper-Based
Model�� 243
13.9 Laboratory Task 2 (Optional): Vessel Sprouting by
Mechanical Stimuli on Paper Model�� 245
14 Advanced Topics�� 249
14.1 Cartilage Tissue Engineering�� 249
14.2 Bone Marrow Transplantation�� 252
14.3 Cardiac Patches �� 253
14.4 Immunoisolated Pancreas �� 255
14.5 Kidney Tissue Engineering �� 256
14.6 Other TE Transplants�� 258
Index�� 261
About the Author
Jeong-Yeol Yoon received his B.S., M.S., and Ph.D. degrees in chemical engineer-
ing from Yonsei University, Seoul (South Korea), in 1992, 1994, and 1999, respec-
tively. His dissertation advisor was Dr. Woo-Sik Kim, and his co-advisor was Dr.
Jung-Hyun Kim, while he worked primarily on polymer colloids. Dr. Yoon received
his second Ph.D. degree in biomedical engineering from the University of California,
Los Angeles, in 2004, working on lab-on-a-chip and biomaterials under the guid-
ance of Dr. Robin L. Garrell. He joined faculty at the University of Arizona in
August 2004 and currently holds split home appointments in the Department of
Biomedical Engineering (primary) and Department of Biosystems Engineering
(secondary). Dr. Yoon also holds joint appointments in Chemistry & Biochemistry
and BIO5 Institute. He is also associate head for graduate affairs in the Department
of Biomedical Engineering, starting from July 2018. He is currently directing
Biosensors Lab. Dr. Yoon is a member of IBE, ASABE, SPIE, BMES, and ACS and
was councillor-at-large for IBE for the 2010 and 2011 calendar years. He was the
president of the Institute of Biological Engineering (IBE) in 2015. Dr. Yoon cur-
rently serves as editor-in-chief for the Journal of Biological Engineering (the offi-
cial journal of IBE), associate editor for Biosensors and Bioelectronics (Elsevier),
and editorial board member for Scientific Reports (Springer Nature) and
Micromachines (MDPI). Dr. Yoon is the sole author of another Springer book,
Introduction to Biosensors – From Electric Circuits to Immunosensors, second edi-
tion, published in 2016, written similarly to this book.
xiii
Chapter 1
Introduction
In this chapter, we will learn several different definitions of tissue engineering as

well as its applications.
Inquiry 1. In your own words, define tissue engineering.
Inquiry 2. What can we do with tissue engineering?
1.1 Narrow Definition of Tissue Engineering
When a tissue or an organ starts malfunctioning and drugs cannot resolve such
problems, you may wish to “substitute” the damaged tissue or organ with a new one.
Tissue or organ can be harvested from a human donor, either deceased or alive, and
transplanted to the patient. This procedure is called organ transplantation. While it
has successfully been practiced in the past, you have probably heard about the
extremely long waiting list of organ transplantation, and many patients would die
before receiving it. Because of this difficulty, synthetic materials have been used to
replace the damaged tissue or organ, made from metals, polymers, or ceramics.
They are called implants, which have shown limited successes in organs, such as
bones. However, such implants cannot metabolize nutrients, cannot produce pro-
teins, etc., and subsequently cannot truly replace the functions of tissues or organs.
Therefore, a new cell-based approach is needed to replicate a living tissue’s or
organ's structure and function. Tissue or organ can be damaged from traumatic
injury, degradation from exercise, cancer, aging, and adult-onset deficiency such as
diabetes. As tissue is made from cells and extracellular matrix (ECM), it is impor-
tant to replicate both the cells and ECM. (Implants may replace the ECM but not the
cells.) Both the cells and ECM must be “engineered” to provide structural integrity
and metabolic behavior found in normal tissue. With the narrow definition of tissue
engineering, engineered mimic of the ECM is constructed, made from either a natu-
ral material (e.g., collagen fibers) or a synthetic material (e.g., polymers). This ECM
© Springer Nature Switzerland AG 2022 1

J.-Y. Yoon, Tissue Engineering, https://doi.org/10.1007/978-3-030-83696-2_1
2 1 Introduction
mimic is called tissue-engineered scaffold (TE scaffold) or just scaffold. Cells are
then harvested from a patient or a donor. We can also use stem cells. They are cul-
tured and seeded on the scaffold in a laboratory, that is, in vitro (meaning “in a
representation,” typically representing a dish, flask, beaker, tube, etc.). The antonym
to in vitro is in vivo, meaning “in life,” typically representing a (human) body.
Sometimes the term ex vivo is used as an antonym to in vivo. While in vitro and ex
vivo do not necessarily represent the same, these two terms are sometimes used
interchangeably. Once the cells proliferate and exhibit normal metabolism in a scaf-
fold, it is then transplanted back to the body to replace the tissue or organ. This final
product is called tissue-engineered transplant (TE transplant). This procedure
describes the narrow definition of tissue engineering.
Early successes in tissue-engineered constructs date back to the late 1990s. The
most famous example would be the work by Cao et al., published in 1997, where a
tissue-engineered human ear was constructed on a nude mouse [Cao et al., 1997;
Bear in mind that there were many limitations and issues with this early pioneering
work]. Since then, tissue engineering has become widely known and practiced. In
the early 2000s, it has been established as a discipline. Tissue engineering is still a
new discipline at the time of writing.
Figure 1.1 shows one example of creating a tissue-engineered transplant (TE
transplant), that is, narrow definition of tissue engineering. Cells are removed from
a healthy host (or a healthy tissue from the same patient). They are cultured in a
laboratory, and the best-performing cell line is isolated. If stem cells are used, they
should also be differentiated using proper differentiation factors, physical stimuli,
and other environmental factors. A scaffold is constructed and the chosen cells are
seeded. Once the transplant is complete, it is transplanted to the patient.
Tissue-engineered transplants have numerous benefits over implants. Many
implants can be rejected from a body via inflammatory and immune responses,
where such issue is minimal with TE transplants. Since the best-performing cell line
can be selected and the scaffold design can be optimized, the resulting TE trans-
plants can perform far better than implants, while there is no availability issue com-
mon in organ transplants.
1.2 arly Attempt in Scaffold Development:

E
Decellularized Matrix
Since its conception in the early 2000s, scaffold design has been considered quite
challenging toward fully mimicking the natural ECM. One early attempt was the
use of natural ECM rather than designing and fabricating a whole new scaffold. An
organ from a dead body (cadaver) was harvested and the cells were removed with
surfactant (decellularization). This decellularized matrix was then used as a scaf-
fold. Ott et al. demonstrated this method using the heart from cadaver (published in
2008). After harvesting, they decellularized with surfactant (Fig. 1.2) and added
1.2 Early Attempt in Scaffold Development: Decellularized Matrix 3
Fig. 1.1 Narrow definition of tissue engineering. (Ude et al., 2018. (C) Open access article distrib-
uted under the terms of the Creative Commons Attribution 4.0 International License)
cardiac cells onto it. Cells were proliferated and maintained for up to 28 days in a
bioreactor (in vitro) (Fig. 1.3). The resulting construct generated the pump function
equivalent to about 2% of adult heart function. While 2% seemed very low, it was
the first demonstration of a thriving TE heart with pump function. It showed the
early promise of tissue engineering and has received a lot of media attention.
This method was groundbreaking in many aspects, eliminating many organ
transplantation issues, especially tissue healthiness or organ availability. However,
it still suffered numerous problems, such as (1) availability of cadavers and (2) lack
of engineering control over the scaffold design and structure. Therefore, follow-up
works have been focused on the design and construction of “engineered” scaffolds
that would meet the requirements of:
–– Size (volume) that can “process” the required amounts of nutrient metabolism,
protein production, waste removal, etc.
–– Mechanical properties that can fit within the available space, resist external
bending and shear force, etc.
–– Longevity that can last for a necessary duration of time
4 1 Introduction
Fig. 1.2 Decellularization of cadaveric heart. (Ott et al., 2008. Reprinted with permission, (C)
2008 Springer Nature)
1.3 Simple TE Transplant Example: Skin
One of the early examples of TE transplants is skin transplant as skin is highly pro-
lific and self-renewable, thus relatively easy to replicate with tissue engineering
technology. The skin has two layers: the outermost and top layer of the epidermis
and the immediate inner layer of the dermis (just underneath the epidermis)
(Fig. 1.4). Keratinocytes and fibroblasts make up most of the epidermis and dermis,
and they proliferate quite well both in vivo and in vitro. Damages of the epidermis
can be healed relatively quickly and do not generally require transplants. However,
severe burns damage the dermis layer and do not heal very well and usually leave
scars, where TE skin transplant can be the right solution. Diabetic patients also have
skin ulcer problems that do not heal, called diabetic ulcers (a famous example is a
diabetic foot), where TE skin transplant can also be utilized. TE skin transplant can
also be used for cosmetic purposes (plastic surgery) to replace the aged skin with a
fresh one.
As skin structure is relatively simple, a simple hydrogel of collagen fibers can be
used as a TE skin scaffold. A gel is a network of natural or synthetic polymers that
are cross-linked, filled with liquid. If the liquid is water, it is specifically called
1.3 Simple TE Transplant Example: Skin 5
Fig. 1.3 Perfusion bioreactor for constructing a tissue-engineered transplant from the decellular-
ized cadaveric heart. (Ott et al., 2008. Reprinted with permission, (C) 2008 Springer Nature)
Fig. 1.4 Human skin. The outermost layer is the epidermis (consisting of squames and keratino-
cytes), followed by the dermis (consisting of fibroblasts and ECM)
6 1 Introduction
Fig. 1.5 Bilayered

tissue-engineered skin
transplant (MyDerm). (Ude
et al., 2018. (C) Open
access article distributed
under the terms of the
Creative Commons
Attribution 4.0
International License)
hydrogel. Hydrogels contain as much as >90% water, which is similar to human

tissues. Human ECM is made from collagen fibers, where the protein collagen is
polymerized and bundled together to form a highly cross-linked fiber network.
Therefore, human ECMs are also hydrogels. Keratinocytes and fibroblasts are
seeded on these collagen gels and proliferated in vitro to create a TE skin transplant.
Two layers are needed, one with keratinocytes to form the epidermis and the other
with fibroblasts to form the dermis (Fig. 1.5). The collagen fibers can be modified
with growth factors, drugs, etc., and can also be engineered to exhibit appropriate
tissue property (elasticity, tensile strength, etc.).
1.4 Simple TE Transplant Example: Pancreas
Another example is the TE pancreas transplant. The pancreas is located just under-
neath the liver, and one of its primary functions is the release of protein insulin,
which regulates the glucose level in a body. When there is a problem with insulin,
such a disease is called diabetes. While drugs and adequate exercise may control the
diabetes symptoms, it is often necessary to self-inject insulin daily, sometimes more
than once a day. While continuous glucose monitoring (CGM) system and subse-
quent automatic insulin injection have become a reality, the ultimate solution would
be the use of TE pancreas transplant. Pancreatic cancer is another severe problem
whose mortality rate remained very high among all cancer types. Again, a TE pan-
creas transplant may be able to resolve this disease.
Decellularized cadaveric pancreas can be used as a TE scaffold, and the β-islet
cells (the primary cells in the pancreas) can be seeded and proliferated in vitro. The
β-islet cells can be harvested from a healthy donor. Unlike organ transplants, the
donor does not need to provide the whole pancreas; a small number of β-islet cells
can be extracted and proliferated in a laboratory (in vitro). The resulting TE
1.4 Simple TE Transplant Example: Pancreas 7
transplant may be rejected in the long run via immune reaction, and therefore, the
immunosuppressant drug should be taken throughout the patient’s lifespan. If the
donor is very well matched with the patient, such immune rejection could be
minimal.
A better alternative is the use of a semipermeable membrane as a TE scaffold. As
shown in Fig. 1.6, many different forms of such semipermeable membranes can be
used: (A) a cylindrical tube (macrocapsule), (B) a push-pull device with refillable
oxygen in it, and (C) a sphere (microcapsule). β-islet cells are loaded inside these
devices, where the semipermeable membrane allows the movements of oxygen,
nutrients, wastes, and protein products (e.g., insulin), moving freely inside and out.
However, these membranes’ pores are small enough to keep the β-islet cells inside
and not allow the immune cells (white blood cells) to attack the β-islet cells. This
procedure is called immunoisolation, which has been a popular method in designing
a TE scaffold.
Fig. 1.6 Immunoisolation methods can be used toward TE pancreas transplant. (Hu & de Vos,
2019 (C). Open access article distributed under the terms of the Creative Commons Attribution 4.0
International License)
8 1 Introduction
1.5 xpanded Definition of Tissue Engineering:

E
Organ-on-a-Chip (OOC)
Another emerging application of tissue engineering is organ-on-a-chip or simply

organ-on-chip (OOC). Organ-on-a-chip (OOC) is a special case of lab-on-a-chip
(LOC). Lab-on-a-chip (LOC) is a miniaturized laboratory fabricated on a “chip.”
Chips are made with semiconductor manufacturing technology, for example, photo-
lithography. In recent years, soft lithography is more popularly used over photoli-
thography. In both methods, chips are made from silicone or silicone-based polymer,
while other polymers can also be used. LOC is essentially a network of wells inter-
connected with sub-millimeter or micrometer-sized channels. The reagents and
specimens are mixed, incubated, reacted, and quantified by various sensors and bio-
sensors in LOCs (Fig. 1.7). LOCs have popularly been used toward point-of-care
clinical diagnostics, field-based sensing and biosensing applications, etc.
There has been a growing interest in adapting these LOCs to replace the in vitro
cell assays toward improved recapitulation of mammalian tissues. For example,
mammalian cells can be added and proliferated within the LOC wells or channels
mimicking the actual tissue structures. These are called organ-on-chips (OOCs).
Hence, this method utilizes the tissue engineering concept, where the LOC is the TE
scaffold. One of the successful demonstrations of OOC is shown in Fig. 1.8, mim-
icking a human lung (thus lung-on-a-chip). A single channel is separated by a mem-
brane, where the lung epithelial cells are seeded and proliferated on it. The bottom
channel represents the blood flow, while the top channel represents the air low. A
single channel can be utilized to mimic the unit lung behavior, or a collection of
channels can be utilized to simulate the overall tissue-level behavior.
Fig. 1.7 Lab-on-a-chip is

a network of channels and
wells fabricated on silicone
or silicone-based polymer.
(Picture was taken by
Vjsiebens in March 2009
and placed in the public
domain. Accessed January
2021 from http://commons.
wikimedia.org/wiki/
File:AutoFISH.jpg)
1.5 Expanded Definition of Tissue Engineering: Organ-on-a-Chip (OOC) 9
Fig. 1.8 Lung-on-a-chip example. (Shrestha et al., 2019. (C) Open access article distributed under
the terms of the Creative Commons Attribution 4.0 International License)
OOCs are typically not for in vivo use. Instead, OOCs provide a laboratory in
vitro platform for the following applications:
–– Cancer study: carcinogenesis (formation of cancer); metastasis (spread of can-
cer), etc.
–– Stem cell differentiation study: identification and optimization of physical and
biological cues to induce successful differentiation to stem cells
–– Biocompatibility test of synthetic materials (e.g., implants)
–– Drug efficacy and toxicity tests
–– Cosmetics toxicity tests
–– Studying the long-term exposure of environmental toxicants to human tissues
and organs
The above studies have conventionally been conducted by (1) in vitro cell assays,
(2) animal tests, and (3) human clinical trials, in this order. There are huge gaps
between (1) and (2) – different responses from individual cells versus tissue or
organ – and between (2) and (3) – species difference between animal and human. As
human cells are used to create a tissue or organ mimic in OOCs, such gaps would be
minimized and eventually eliminated. Besides, as the final device does not need to
be transplanted back to humans while there is a huge commercial potential, OOCs
have gained substantial popularity in tissue engineering research.
10 1 Introduction
1.6 Overview of This Book
This book is intended for the audience with limited understanding and experience in
wet laboratory-based biology. Brief and easy-to-practice laboratory modules are
introduced in each chapter. They can certainly be used for an undergraduate-level
class (or a graduate-level introductory class) with lecture and laboratory compo-
nents. However, it will equally be useful for the course with no laboratory compo-
nents. The laboratory procedures are well-documented, with ample figures,
photographs, experimental data, and subsequent analyses, which provides the vir-
tual laboratory experience. It will equally be great for the people self-studying tis-
sue engineering.
Chapter 2 will learn the fundamental basics of cell culture and the basic labora-
tory skills necessary to conduct it. Bacterial cell culture (the easiest cell culture)
laboratory will be introduced, and the use of a microscope will be demonstrated.
Chapter 3 will then advance to cell metabolism – how cells consume nutrients
and oxygen and generate wastes. Feeding and passaging – the two most essential
skills for mammalian (including human) cell culture – will be demonstrated as labo-
ratory exercises.
In Chapter 4, once cells are successfully cultured, it is crucial to “stain” the dif-
ferent areas of cells and image them. This staining and imaging process is mostly
done by fluorescence microscopy. Basic principles of fluorescence, staining, and
fluorescence microscopy will be covered, along with the laboratory exercise on
fluorescence cell imaging.
Chapter 5 will learn stem cells, which are quite crucial for many tissue engineer-
ing applications (although not always necessary). Stem cell culture and confirma-
tion of differentiation will be demonstrated as laboratory exercises.
Chapter 6 will learn various biomaterial surfaces – basically what material is
preferred for tissue engineering applications and whether a surface modification is
necessary or not. As tissue engineering requires the use of “engineered” scaffold
(including organ-on-a-chip), choice and modification of biomaterial surface must
be preceded before constructing the scaffold. Several different biomaterial surfaces
will be fabricated or prepared in the laboratory exercise. Their surface properties,
especially the two most popularly used ones – contact angle and surface rough-
ness – will be evaluated through the laboratory exercise.
Chapter 7 will learn the focal adhesion. It is a primary mechanism for most mam-
malian (including human) cells to be anchored on the extracellular matrix (ECM) to
form a functional tissue, which must be duplicated in tissue-engineered devices.
Fluorescence imaging to confirm focal adhesion will be exercised through a labora-
tory exercise.
Chapter 8 will learn contact guidance. Through focal adhesion, cells can be
aligned with the shape and pattern of ECM, which is the definition of contact guid-
ance. Again, contact guidance must be demonstrated in tissue-engineered devices to
1.6 Overview of This Book 11
create a fully functioning tissue-engineered transplant or tissue/organ mimic (OOC).

Contact guidance on a simple microgroove will be demonstrated as a laboratory
exercise.
In Chapter 9, two popular methods of creating 3D TE scaffold will be explained –
electrospinning and 3D printing. Laboratory exercises will be introduced on these
two methods.
In Chapter 10, the engineering approach for designing in vitro cell culture and
bioreactor will be introduced. Characteristic times will be introduced to design the
in vitro culture, including the initial number of cells, time for culture, consideration
of Hayflick limit, the volume of a bioreactor, consumption rates of nutrients and
oxygen, and the design of TE scaffold based on diffusional lengths, etc. Various
types of bioreactors for tissue engineering will also be covered.
In Chapter 11, organ-on-a-chip will be discussed as the first representative appli-
cation of tissue engineering. Laboratory exercises on organ-on-a-chip fabrication
and operation will be introduced.
In Chapter 12, TE skin transplant will be discussed as the second representative
application of tissue engineering. A quick laboratory exercise of TE skin transplant
will be demonstrated.
In Chapter 13, angiogenesis (formation of a new blood vessel) will be introduced
toward vascularization as it is necessary to create a fully functioning TE device.
Angiogenesis-on-a-chip will be demonstrated as its laboratory example.
In Chapter 14, advanced topics will be covered, including cartilage tissue engi-
neering, bone marrow transplantation, cardiac patches, immunoisolated pancreas,
and kidney tissue engineering.
Figure 1.9 graphically summarizes the overall structure of this book.
Review Questions
1. Compare narrow and broader definitions of tissue engineering (TE).
2. What is extracellular matrix (ECM)?
3. What is TE scaffold?
4. What is hydrogel?
5. What is decellularization? How is it used for tissue engineering applications?
6. Injecting β-islet cells from a well-matched donor to a diabetic patient provides
only temporary relief. Why? How can you resolve this issue?
7. What is immunoisolation, and why is it necessary for tissue engineering?
8. How is TE skin transplant made?
9. What are the benefits of organ-on-a-chip (OOC)?
12 1 Introduction
Fig. 1.9 Overview of this book
References
Cao, Y., Vacanti, J. P., Paige, K. T., Upton, J., & Vacanti, C. A. (1997). Transplantation of chon-
drocytes utilizing a polymer-cell construct to produce tissue-engineered cartilage in the
shape of a human ear. Plastic and Reconstructive Surgery, 100(2), 297–302. https://doi.
org/10.1097/00006534-199708000-00001
Hu, S., & de Vos, P. (2019). Polymeric approaches to reduce tissue responses against devices
applied for islet-cell encapsulation. Frontiers in Bioengineering and Biotechnology., 7, 134.
https://doi.org/10.3389/fbioe.2019.00134
Ott, H. C., Matthiesen, T. S., Goh, S. K., Black, L. D., Kren, S. M., Netoff, T. I., & Taylor,
D. A. (2008). Perfusion-decellularized matrix: Using nature’s platform to engineer a bioartifi-
cial heart. Nature Medicine, 14, 213–221. https://doi.org/10.1038/nm1684
Shrestha, J., Ghadiri, M., Shanmugavel, M., Bazaz, S. R., Vasilescu, S., Ding, L., & Warkiani,
M. E. (2019). A rapidly prototyped lung-on-a-chip model using 3D-printed molds. Organs-on-
a-Chip, 1, 100001. https://doi.org/10.1016/j.ooc.2020.100001
Ude, C. C., Miskon, A., Idrus, R. B. H., & Bakar, M. B. A. (2018). Application of stem cells in tis-
sue engineering for defense medicine. Military Medical Research, 5, 7. https://doi.org/10.1186/
s40779-018-0154-9
Chapter 2
Cell Culture
In the previous chapter, we have learned the overview of tissue engineering princi-
ples and their applications. All tissue engineering applications start from growing
cells in an appropriate culture, which is the first topic that we should learn. Here are
two inquires for you:
Inquiry 1. What is cell culture?
More importantly, how do we “feed” cells and maintain their health? How can
we assess their health (typically by monitoring their shape, metabolism, and protein
production)?
Inquiry 2. How can we classify cell types?
Different cell types require varied methods of cell culture. Have you heard
anchorage-dependent versus anchorage-independent cells? Have you heard primary
versus stem cells? How about transformed or immortalized cells?
2.1 What Is Cell Culture?
While there are many different definitions of cell culture, we can always find the
following three components in its definition:
(1) In vitro, meaning “in a representation,” that is, in a tube or a culture dish, and
outside a living organism (antonym is in vivo)
(2) Maintain cells in a viable state, indicating that cells should be alive and actively
proliferating (dividing or reproducing)
(3) Maintain cells in a metabolically active state
To accomplish all three, we need to feed oxygen, nutrients, and growth factors
appropriately. Maintenance of metabolically active state is quite crucial for tissue
engineering applications. Cells should produce necessary proteins and respond to

14 2 Cell Culture
environmental factors (e.g., physical and biological cues) to perform essential func-
tions toward tissue engineering applications.
Cells are typically harvested from humans for most tissue engineering applica-
tions. For specific tissue engineering applications, we can also use the cells from
animals, plants, and bacteria. In many tissue engineering applications, it may be
preferable to use a cell population descended from a single cell, referred to as a cell
line, as they would contain identical genetic information. However, most mammal
and human cells would divide only up to a certain number of doublings (known as
Hayflick limit, discussed later in Sect. 10.2), and there are limitations in using cell
lines. On the other hand, certain cells can divide into an indefinite number of dou-
blings, appropriate as cell lines. These cells are referred to as immortalized cells.
Cancer cells are probably the most well-known example of immortalized cells (they
are spontaneously immortalized cells). One of the earliest human cell lines is HeLa
cells, descended from the cancer cell from Henrietta Lacks in the 1960s, who died
of cancer. Figure 2.1 shows the microscopic image of cultured HeLa cells with
Hoechst staining, which stains the cell nuclei in blue. (These days, cell nuclei are
stained with better dye, DAPI, which will be discussed later in Sect. 4.3.)
Cell cultures are commonly used for various applications. From the definition of
cell culture, cells must maintain a metabolically active state in cell culture, produc-
ing adequate amounts of proteins and responding to various environmental factors.
The protein production feature can be utilized in many different industrial applica-
tions. Such proteins are typically used as drugs, including vaccines. A large-scale
bioreactor can also be used instead of a simple culture dish to produce many such
drugs. Such specific application is often referred to as cellular engineering, which
shares many standard features with tissue engineering.
Protein products from cell culture include (1) enzymes, (2) hormones, (3) viral
vaccines, (4) antibodies, and (5) cytokines. Enzymes are proteins that function as
biological catalysts, that is, facilitating and speeding up chemical reactions. Many
of them can be utilized as drugs to facilitate or speed up specific chemical reactions.
Fig. 2.1 HeLa cells with

Hoechst staining. (The
picture was taken by Masur
in January 2007 and placed
in the public domain.
Accessed January 2021
from https://commons.
wikimedia.org/wiki/
File:HeLa_cells_stained_
with_Hoechst_33258.jpg)
2.2 Cell Physiology: Cell Membrane and Cytoskeleton 15
Hormones are not necessarily proteins but are produced by the cells in certain
glands, regulating physiology and behavior. Viral vaccines are typically attenuated
or modified viruses that can trigger human immune responses, providing acquired
immunity but not making people sick. Recombinant protein and messenger RNA
(mRNA) are also used as viral vaccines, as seen from the recent vaccine develop-
ment for COVID-19. While vaccines to nonviral pathogens also exist, viral vaccines
are by far the most important ones. Antibodies are the proteins secreted by B lym-
phocytes (or B cells in short) in the blood that make up the critical part of humans’
and mammals’ immune systems. They bind to specific antigens (proteins, viruses,
bacteria, etc.) and neutralize their action. While antibodies have previously been
used for diagnostics and biosensing applications in the past, they are now more
frequently used in treating diseases, including many different autoimmune diseases
and various types of cancer. Cytokine (cyto = cell and kine = movement) is a collec-
tive term for the small proteins secreted from cells used for cell signaling purposes.
Specific cytokines can signal the onset or suppression of certain diseases and are
subsequently used as drugs. Famous examples include interferons (used to treat
cancer) and interleukins (secreted by inflammatory and immune cells).
Cell culture is also being popularly used for evaluating the efficacy and toxicity
of drugs. Once a new drug is identified or developed, it is assessed for its efficacy
and toxicity in three different steps, namely, (1) in vitro test, (2) animal test, and (3)
human clinical trial. Cell culture has frequently been used for the first step – in vitro
test. In such a test, varying amounts (doses) of the drugs are added to the cell cul-
ture, for example, human liver cells, human kidney cells, etc., and the cells’ viability
and metabolic status are assessed. The number of live versus dead cells is the most
frequently evaluated after a certain drug exposure time (ranging from a couple of
hours to a few weeks). The shape of cells (cell morphology) can also be assessed.
Production of specific proteins (e.g., albumin from liver cells) is also frequently
evaluated. While a simple 2D cell culture has widely been investigated for drug
efficacy and drug toxicity tests, a more sophisticated 3D cell culture has emerged as
an improved alternative. While 3D culture provides an environment that resembles
the in vivo human environment, it still lacks a network of vessels in most organs in
the human body. Organ-on-a-chip (OOC) is essentially a 3D culture with vessel
structures (thus with the active fluid flow) that can better mimic the human organ.
Most tissue engineering technologies are used in developing and using OOCs. OOC
has already been explained in Chap. 1 briefly, will be frequently mentioned through-
out this book, and discussed in detail in Chap. 11.
2.2 Cell Physiology: Cell Membrane and Cytoskeleton
Before we tackle cell culture, it is essential to learn the basics of cell physiology.
Two components of cells are critical in learning cell culture and tissue engineering:
(1) membrane and (2) cytoskeleton. Here is why: most cells used in tissue engineer-
ing applications are anchorage-dependent, that is, they should be anchored on a
16 2 Cell Culture
surface to behave normally. (Do not worry – we will cover the difference between
anchorage-dependent and anchorage-independent cells later in this chapter.) And
cells’ anchorage to a surface is governed by their membrane and cytoskeleton.
The cell membrane is made from a phospholipid bilayer. Each layer is made
from phospholipid, where the head is made from a phosphate group (PO3−) that is
negatively charged. The tail of this phospholipid is simply lipid (= a long hydrocar-
bon chain). Such hydrocarbon chain is neither charged nor polar, thus insoluble to
water (water is strongly polar and thus a strong solvent). Therefore, this lipid tail is
hydrophobic (hydro = water and phobic = hating; sometimes referred to as lipo-
philic although less frequently used), while the phosphate head is hydrophilic (philic
= loving) as it is negatively charged. As the majority mass of a cell and its environ-
ment is water, the lipid tails should associate with each other to avoid the water
molecules’ entropy penalty. Such association is referred to as hydrophobic interac-
tion. This self-assembly will form a bilayer of phospholipid molecules, which is the
cell membrane’s very nature, as shown in Fig. 2.2.
As you can see from Fig. 2.2, the inner layer of a cell membrane is hydrophobic,
while the outer surfaces (one at the outside of a cell and the other facing the inside)
are negatively charged (and thus hydrophilic). In the cell membrane, there are mem-
brane proteins. Proteins are polymers of amino acids, where some amino acids are
hydrophobic and other amino acids are hydrophilic. Depending on the amino acid
sequence, some parts of a protein are hydrophobic, while the others are hydrophilic.
The hydrophobic portion of a membrane protein is associated with a phospholipid
bilayer’s lipid portion and forcing the membrane protein to stay within a phospho-
lipid bilayer. The hydrophilic part of a membrane protein is exposed to a cell mem-
brane’s outer or inner side, as shown in Fig. 2.2. They communicate with the
Fig. 2.2 Cell membrane. Phospholipids are colored in blue, showing their hydrophilic and nega-
tively charged heads and hydrophobic tails. They are associated in a bilayer form, making up a cell
membrane. Membrane proteins, colored in orange, are “floating” on this cell membrane, extruding
their structure to both inside and outside the cell (left) or only outside the cell (right)
2.2 Cell Physiology: Cell Membrane and Cytoskeleton 17
molecules outside the cells (extracellular matrix) or inside the cells (mostly cyto-
skeletons, which will be discussed in the following section). Specific membrane
proteins play critical roles in cell–cell or cell–surface adhesion, which will be dis-
cussed later in this chapter.
Cytoskeleton means the bone (= skeleton) in a cell (= cyto). It consists of three
components: (1) actin filaments, (2) microtubules, and (3) intermediate filaments.
Actin filaments are two-stranded helical polymers made from the protein actin.
They have flexible structures of diameter 5–9 nm. As shown in Fig. 2.3, they are
most highly organized on the periphery of a cell, while the actin fibers that cross a
cell (stress fibers) can also be found. Actins are initially formed as G-actin, where
“G” stands for “globular,” that is, rounded shape. Polymerization of G-actins leads
to F-actin, where “F” stands for “filament.” This polymerization (growth) proceeds
primarily in one direction, with a fast-growing “+” end. This polymerization rate
varies by the cell activity, that is, whether the cells are actively proliferating (divid-
ing), moving, or quiescent (neither proliferating nor moving). For the cells actively
proliferating and/or moving, the mean filament lifetime is relatively short (8 min),
with a small fraction (40%) of actins polymerized. For the quiescent cells, that is,
anchorage-dependent cells occupying most of the surface (= confluent monolayer,
discussed in the next section), the mean filament lifetime is quite long (40 min) with
a large fraction (70%) of actins polymerized. Actin filaments play a crucial role in
cell adhesion to a surface for anchorage-dependent cells.
Microtubules are hollow cylinders comprising the protein tubulin. Their outer
diameter is 25 nm, much larger than that of actin filaments. They are also much
more rigid and straight than actin filaments due to their cylindrical, tubular struc-
ture. Microtubules are usually attached to a single microtubule-organizing center, as
shown in Fig. 2.3. Like actin filaments, microtubules are polymerized from tubulins,
and this polymerization proceeds in one direction, with a fast-growing “+” end.
Microtubules play vital roles in the structure of cilia and flagella of cells, which
provides rigidity and assists in generating cellular motion and cell division through
forming mitotic spindle.
Fig. 2.3 Actin filaments (left), microtubules (middle), and intermediate filaments (right)
18 2 Cell Culture
Intermediate filaments are rope-like fibers made from a heterogeneous family of

proteins. They are quite long, with a diameter of around 10 nm. They form a mesh-
work across the cell, providing mechanical strength.
2.3 Focal Adhesion
As mentioned, actin filaments are the most critical cytoskeleton in cell culture,
involved in cell adhesion to a surface. For the cells in mammals and humans, that is,
cells in vivo, such surface is extracellular matrix (ECM). ECM provides a surface
where anchorage-dependent cells can be anchored to and give a specific structure
built into tissue and ultimately an organ. In cell culture, it is essential to provide an
artificial surface that mimics such ECM. The most desirable cell adhesion to a sur-
face is focal adhesion (for anchorage-dependent cells), and its schematic is shown
in Fig. 2.4. In focal adhesion, certain proteins in ECM or a specific structure and
character of an artificial surface can be recognized by a membrane protein integrin.
Such binding to integrin triggers adaptor proteins’ binding within the cell, including
actinin, talin, tensin, paxillin, and vinculin. Vinculin is the essential protein that can
connect integrin and actin filaments. While other adaptor proteins may or may not
be found at the focal adhesion site, you can always find vinculin. Identification of
vinculin has been used as a preferred method of confirming focal adhesion of
anchorage-dependent cells. In this manner, a continuous link is formed from a sur-
face, cell membrane (integrin), to the cytoskeleton (actin filaments), providing quite
Fig. 2.4 Focal adhesion of an anchorage-dependent cell to a surface

2.5 Cell Classification: Normal Versus Immortalized Cells 19
a tight adhesion of cells to a surface. Focal adhesion will be further discussed in

Chap. 7.
2.4 Cell Classification: Anchorage-Dependent Versus

Anchorage-Independent Cells
At this point, you will probably have a good idea of the difference between
anchorage-dependent and anchorage-independent cells. Anchorage-dependent cells
require a surface they can anchor. The surface is typically extracellular matrix
(ECM) in vivo, while an artificial surface can also be used for the cell culture in
vitro. Anchorage-dependent cells cannot be stacked in multilayers on a surface, that
is, they can only form a monolayer. When a surface is saturated with anchorage-
dependent cells in a monolayer (caused by contact inhibition), such condition is
referred to as confluency, for example, 80% confluency represents 80% of the sur-
face is covered by anchorage-dependent cells.
On the other hand, anchorage-independent cells do not require a surface for their
proliferation and metabolism and can be cultured in a suspension. The best exam-
ples are blood cells, including red blood cells (RBCs) and white blood cells (WBCs).
As you can imagine, it will be quite problematic if blood cells are anchored to a
surface (in this case, the wall of blood vessels) – they should be able to move along
the blood flow to circulate throughout the body.
Anchorage-dependent cells can lose their anchorage-dependency and become
anchorage-independent. A famous example is cancer cells – where the normal cells
become “crazy,” dividing indefinitely (no Hayflick limit), not fulfilling their assigned
duties, consuming a large number of nutrients and oxygen, and converting the
nearby cells into cancer cells. Sounds familiar? Yes, zombies! All those zombie
movies are inspired by these crazy cancer cells, although such mutations do not
occur that quickly. As cancer cells are not doing their original duties and forgetting
who they were, they gradually forget their anchorage-dependency (Fig. 2.5).
2.5 Cell Classification: Normal Versus Immortalized Cells
Cells can also be classified based on their growth behavior, that is, normal versus
immortalized. Normal cells can proliferate (divide) for a limited number of times
(typically limited by Hayflick limit; discussed later in Sec. 10.2) and are highly dif-
ferentiated, that is, they perform specific functions. They usually require a variety of
growth factors and other components in the growth media, that is, a serum is pre-
ferred as growth media, which will be discussed in the later section of this chapter.
On the other hand, immortalized cells can proliferate (divide) for an indefinite
number of times, thus immortal. They are sometimes referred to as a more generic
20 2 Cell Culture
Fig. 2.5 Top: Chinese

hamster ovary (CHO) cells
representing anchorage-
dependent cells. The
picture was taken by
Alcibiades in February
2006 and placed in the
public domain (Accessed
January 2021 from https://
commons.wikimedia.org/
wiki/File:Cho_cells_
adherend2.jpg). Bottom:
human neutrophils (more
giant cells with multiple
nuclei within cells; a part
of human white blood
cells) shown together with
human red blood cells
(smaller, donut-like cells),
representing anchorage-
independent cells. The
picture was taken by Dr.
Graham Bears in August
2012 and placed in the
public domain. (Accessed
January 2021 from https://
commons.wikimedia.org/
wiki/File:Neutrophils.jpg)
term of transformed cells, although these two terms’ specific meanings may slightly
differ. Cancer cells are considered as spontaneously immortalized cells. If the
immortalized cells were originally anchorage-dependent cells, they lose their
anchorage-dependency in full or in part and become (partly) anchorage-independent.
Once they lose their anchorage-dependency, they can grow in suspension or multi-
layers without contact inhibition. As they tend to lose their differentiation, it is
essential to retain some differentiation if you plan to use immortalized cells for tis-
sue engineering applications. As they are “crazy” cells, they are not picky eaters and
may not need sophisticated serum as their growth media.
As discussed earlier in this chapter, a healthy cell culture requires (1) a viable
state and (2) a metabolically active state. As immortalized cells focus on the first
condition of a viable state, they comprise the second condition of the metabolically
active state. Therefore, their metabolism, protein production (including cell signal-
ing), and response to environmental factors (including anchorage-dependency) can
be compromised in part or in full. Besides, the rate of DNA mutation increases, and
it becomes harder to fix it.
2.5 Cell Classification: Normal Versus Immortalized Cells 21
Various methods can be used to immortalize normal cells. One of the standard
techniques is infecting normal mammalian cells (= human and mammal cells) with
a virus that can cause cancer. SV-40 (simian vacuolating virus 40 or simian virus 40
in short) is a good example, which is a small DNA virus found in monkeys and
humans (thus “simian”) and can cause cancer to them. Various mammalian cells
have been infected with SV-40, causing them to partially or fully lose their serum
requirement, anchorage-dependency, and thus gaining immortality.
Primary cells and cell lines are often used in place of normal versus immortal-
ized cells. Primary cells are harvested from mammals or humans, and they are most
likely normal cells, that is, differentiated cells. On the other hand, cell lines are the
ones that descended from a single cell and thus have been passaged for a long time
(refer to the next section of cell passaging). While both normal and immortalized
cells can be used for cell lines, cell lines are most likely immortalized cells as they
can proliferate (divide) for an indefinite number of times. In this sense, normal ver-
sus immortalized cells and primary cells versus cell lines are sometimes used inter-
changeably, although their exact meanings differ.
Both primary cells and cell lines should be stored frozen for long-term storage.
Most refrigerators have a dedicated section for freezing, and exclusive freezers are
also available. However, these freezers can only provide a temperature around
−20°C and are inappropriate for storing mammalian cells for the long term. Because
of this, deep freezers are available, which can provide −40 °C or −80 °C, respec-
tively (Fig. 2.6, left). Of course, the −80 °C deep freezer is substantially more
expensive than the −40 °C one. However, the ultimate solution is to store these cells
in liquid nitrogen storage, which will provide −196 °C (the boiling temperature of
liquid nitrogen) (Fig. 2.6, right).
Fig. 2.6 −40 °C deep freezer (left) and liquid nitrogen tank (right) for storing mammalian cells
22 2 Cell Culture
2.6 Cell Classification: Normal Versus Stem Cells
In the previous section, we briefly discussed differentiation. All cells in a human

body retain identical genetic information, and they originate from a single type of
cell (zygote = fertilized egg). In this sense, it is totipotent (toti = all; potent = ability)
stem cell. You can also find stem cells in an adult human body that can differentiate
into a large number of cell types but not a whole organism, called pluripotent stem
cells, or a limited number of cell types, called multipotent stem cells. Examples
include mesenchymal stem cells and hematopoietic stem cells. These stem cells will
be discussed in Chap. 5.
Stem cells are undifferentiated and cannot perform specific functions, while nor-
mal cells are fully differentiated and perform particular operations. Stem cells can
also proliferate (divide) for a small number of doublings or are close to immortal.
However, stem cells are fundamentally different from immortalized cells (including
cancer cells) as they are not “crazy” cells.
In tissue engineering applications, normal cells may not be ideal as they can
proliferate only for a limited number of doublings and require specific growth media
like a serum. The use of immortalized cells may cause several issues, as they are
“crazy” cells and sometimes challenging to retain their functionality. The use of
stem cells is ideal as they can proliferate for a great number of doublings and are not
“crazy” cells. Differentiation can be induced later to make the cells perform specific
duties. However, such differentiation is a relatively complex and delicate process,
and the full and exact mechanism of differentiation is often unknown.
2.7 Maintaining Sterile Environment: Biosafety Cabinet
Cell culture can be contaminated by bacteria or yeast, which is quite a common

problem. In such a case, mammal and human cells (= mammalian cells) compete
with bacteria and yeast for nutrients. Mammalian cells may quickly lose such com-
petition, and the entire cell culture is overrun by bacteria or yeast. Maintaining a
sterile environment is mandatory to avoid such contamination. Preparation of cells
and culture media and in-culture practices (feeding and passaging, which will be
discussed in Chap. 3) should be conducted in a biosafety cabinet, also known as
laminar flow hood. A typical biosafety cabinet is shown in Fig. 2.7, which is a class
II biosafety cabinet.
Class I biosafety cabinet protects the laboratory and personnel, although it does
not provide a sterile environment within the cabinet. Class II biosafety cabinet is the
most common. It protects the laboratory and personnel (class I’s function) and addi-
tionally provides a sterile environment within the cabinet. Class III biosafety cabi-
net protects the laboratory and personnel (class I’s function), provides a sterile
environment within the cabinet (class II’s function), and provides additional protec-
tion for high-risk biological agents.
2.8 Maintaining Sterile Environment: Autoclave 23
Fig. 2.7 A class II biosafety cabinet: (left) overall view and (right) inside view
The core components of a biosafety cabinet are (1) a high-efficiency particulate

air (HEPA) filter and (2) an ultraviolet (UV) lamp. Both are typically located at the
top ceiling of the inside cabinet. HEPA filter removes particulates and aerosols and
must be replaced periodically. UV lamp kills bacteria, yeast, etc., that is, germicidal.
Figure 2.7 (right) was taken during the active experiments, with tubes, tube racks,
pipette tips, pipette tip racks, micropipettes, disposable pipettes, etc. All of them
should be removed after experiments are finished, and the inside surfaces must be
disinfected using bleach solution followed by ethanol. Most biosafety cabinets are
equipped with power outlets connected to the AC power outlets, allowing small
AC-powered equipment such as vortex mixer, rock shaker, mini-centrifuge, etc.
They are also equipped with connectors that can be connected to nitrogen or com-
pressed air tank and a vacuum pump. A vacuum is handy for removing the superna-
tants after centrifuging.
2.8 Maintaining Sterile Environment: Autoclave
An autoclave is another essential piece of equipment for maintaining a sterile labo-

ratory environment. Tubes, transfer pipettes, pipette tips, glassware, etc., can also be
contaminated with bacteria or yeast and must be disinfected. As many of these items
are disposable, that is, for single use, a large quantity of them should be disinfected.
Their smaller size and large amount make them difficult to be disinfected. An auto-
clave is quite useful to disinfect them altogether. As shown in Fig. 2.8, these tubes
24 2 Cell Culture
Fig. 2.8 Autoclave
and tips can be placed inside an autoclave, and high temperature (over 120 °C) and
high pressure (above 15 PSI = 0.1 MPa) are applied to disinfect (= kill) any bacteria,
yeast, etc.
2.9 Cell Culture: CO2 Incubator
Cell culture can be conducted in either an incubator or a bioreactor. While a sophis-

ticated bioreactor is almost mandatory for culturing cells in a large quantity and a
continuous and automated manner, an incubator can also be conveniently used for
small-scale cell culture. For most tissue engineering applications, both incubator
and bioreactor are necessary, as shown in Chap. 1 and Fig. 1.2. Specifically, if many
different cell culture conditions are required for optimizing cell culture parameters,
an incubator becomes essential.
Incubator provides an environment that is optimum for cell culture. It typically
provides a fixed temperature, for example, 37 °C (human’s body temperature), opti-
mum relative humidity (RH), for example, at 95% (preventing water evaporation),
and HEPA-filtered air (providing oxygen but not particulates and aerosols). For cul-
turing mammal or human cells, that is, mammalian cells, a specific incubator type
is necessary, that is, CO2 incubator. As its name indicates, it provides an additional
environmental condition of CO2, typically at 5%. This 5% CO2 is the physiological
condition of most mammalian tissues, allowing them to maintain appropriate pH
2.10 Cell Imaging: Fluorescence Microscope 25
Fig. 2.9 Left: double-stacked CO2 incubator with a CO2 gas tank attached to it; right: multiple cell
culture flasks within a CO2 incubator
(when dissolved into water, CO2 turns into bicarbonate and becomes acidic).
Traditional incubators, for example, without CO2, can be used to culture bacteria.
Figure 2.9 shows a typical CO2 incubator. In this case, two incubators are stacked.
You can see a CO2 gas tank next to it. Within the CO2 incubator, multiple cell culture
flasks are stacked, each being cultured with different cells and varying media
conditions.
2.10 Cell Imaging: Fluorescence Microscope
Once cells are properly cultured, you may wish to image them to count their num-
ber, check their shape (morphology), and identify subcellular components. As cells
are too small to be imaged by a conventional camera, a microscope becomes neces-
sary. A typical microscope is shown in Fig. 2.10, where an objective lens is located
underneath the sample stage. This setup is known as an inverted microscope, which
is popular in cell imaging as it provides more room to a user. When the objective
lens is located on top of the sample stage, it is known as an upright microscope,
which is more traditional but less popular in cell imaging as it does not provide suf-
ficient room to a user.
In cell imaging, it is quite common to stain different subcellular components
with varying fluorescent dyes. It is quite challenging to figure out subcellular parts
purely based on their shapes. For example, nuclei can be stained with a
26 2 Cell Culture
Fig. 2.10 LB (left) and lyophilized powder of E. coli K12 (right)
blue-fluorescent dye, actin filaments with a green-fluorescent dye, and mitochon-

dria with a red-fluorescent dye. As it is not possible to excite all three fluorescent
dyes together, a user needs to excite the fluorescent dyes one by one and acquires
three different fluorescent images. These three images can be stacked together to
create a single image, and of course, such an image is not something you can see
through an eyepiece with your naked eye. More details on cell imaging and fluores-
cence staining are discussed in Chap. 4.
2.11 Laboratory Task 1: Bacterial Cell Culture
While mammalian cells are cultured in tissue engineering applications, we will

learn the basics of cell culture with bacteria in this task. They are very easy to cul-
ture and less prone to contamination (bacteria themselves are contaminants to mam-
malian cell culture!).
Objective 1. Prepare Cells and Media
In this case, we will use lyophilized (= freeze-dried; water is evaporated at low tem-
perature using extremely low pressure, i.e., vacuum, to avoid killing bacteria) pow-
der of Escherichia coli K12, the safe strain of E. coli. We will use LB (lysogeny
broth) as the media for E. coli K12.
1. Wear gloves and a lab coat. The following procedure should preferably be con-
ducted in a biosafety cabinet.
2. Pipette 5 mL of LB into a 15-mL tube (Fig. 2.10, left).
3. Using a 1-mL pipette tip, scoop a tiny bit of lyophilized E. coli K12 and place it
in a 15-mL tube (Fig. 2.10, right). E. coli powder should barely cover the
pipette tip.
2.11 Laboratory Task 1: Bacterial Cell Culture 27
4. Close the lid of a tube and shake it gently using your hand (Fig. 2.11, left).
Objective 2. Culturing in an Incubator
5. Place the tube in an incubator (Fig. 2.11, right). A regular incubator is preferred.
It may be possible to use a CO2 incubator without a CO2 supply. However, it may
contaminate the CO2 incubator and should be used with caution, and the inside
cabinet should thoroughly be disinfected after the culture.
6. Maintain 37 °C and culture for 6 h or overnight (~12 h) (Fig. 2.12).
Fig. 2.11 After gentle mixing of LB and E. coli K12 powder (left), tubes are placed in an incuba-
tor at 37 °C (right)
Fig. 2.12 E. coli suspension culture over time, from 2 h (left), 4 h (middle left), 6 h (middle right),
and overnight (~12 h; right). You can observe more gas formation in the early phase (= active
metabolism) and more turbid solution in the later phase (= a great number of E. coli cells)
28 2 Cell Culture
Objective 3. Spectrophotometric Quantification

The number of E. coli cells can be quantified using spectrophotometry (Fig. 2.13).
As E. coli cells absorb and scatter incoming light, absorbance can be correlated to
the E. coli concentration. Maximum absorbance can be observed in the visible spec-
trum’s red color, for example, from 550 to 750 nm, any wavelength in this region
can be utilized (Fig. 2.14). We will use 600 nm in this case.
Fig. 2.13 Left: LB (as a blank) or E. coli culture suspension is loaded into a cuvette; right: the
cuvette with the sample is placed within a spectrophotometer (a miniature spectrometer with a
light source and a cuvette holder from Ocean Optics)
Fig. 2.14 Intensity spectrum of E. coli suspension. Note that high-intensity (I) value is correlated
to smaller absorbance (A), as A = −log I / I0
2.11 Laboratory Task 1: Bacterial Cell Culture 29
7. Dispense 0.5 mL of LB into a disposable micro-cuvette (path length of 1 cm)

(Fig. 2.13, left). Set this as a blank for a spectrophotometer. (You can also set
water as a blank and subtract this LB solution’s absorbance from all absorbance
measurements.) Wipe the exterior of each cuvette with KimWipes (delicate task
wipes), if necessary. Discard the micro-cuvette after each use. If a larger volume
is needed for disposable cuvettes, increase the volume of LB solution and tube
size in step 2 as needed.
8. Take 0.5 mL of E. coli culture at 0 h, 2 h, 4 h, 6 h, and 12 h (overnight – optional).
If sediments (precipitates) are visible at the bottom of the tube, do not disturb
them and draw from the supernatant. Measure absorbance (A) at 600 nm using a
spectrophotometer (Fig. 2.13, right). If the spectrometer is calibrated with the
blank solution (step 7), it will provide absorbance (A) values. If not, it will pro-
vide intensity (I) values. If this is the case, use the equation A = −log I / I0, where
I0 is the intensity of blank solution (in this case LB), and I is the intensity of your
bacterial suspensions.
9. Plot the absorbance against time (Fig. 2.15).
The result shown in Fig. 2.15 follows the classic cell growth kinetics shown in
Fig. 2.16 quite well. The first couple of hours correspond to the exponential growth
phase. After 4 h, the growth starts to slow down and is plateauing, corresponding to
the stationary phase. While this curve was constructed from bacterial cell culture,
similar behavior can be observed with mammalian cells. More details on cell growth
kinetics will be covered in Chap. 3.
While a simple spectrophotometric measurement was used in this laboratory
exercise, plating and colony counting is a preferred bacterial quantification method.
It is more accurate, accounting only for the viable cells. However, we will not cover
this method in this chapter as it is not relevant to mammalian cell culture.
Fig. 2.15 Absorbance values at 600 nm for E. coli K12 culture are plotted against time
30 2 Cell Culture
Fig. 2.16 Cell growth

kinetics
2.12 aboratory Task 2: Fluorescence Microscopic Imaging

L
of Mammalian Cells
While we have a dedicated chapter on this topic, it will be useful to familiarize

yourself with the use of a fluorescence microscope (Fig. 2.17).
10. Purchase a prepared slide of mammalian cells, for example, FluoCells Prepared
Microscope Slides from Invitrogen (part of ThermoFisher Scientific).
11. Secure three different filter cubes for three fluorescent dyes. For example, cell
nuclei are typically stained with DAPI (functioning as both bioreceptor for
nuclei and blue-fluorescent dye). A DAPI filter cube should be installed in one
slot of the filter cube holder. Filter cube can also be selected based on the exci-
tation and emission wavelengths of the fluorescent dye. Likewise, actin fila-
ments are typically stained with FITC-phalloidin, where phalloidin is a receptor
binding to actin filaments, and FITC is a green-fluorescent dye. Mitochondria
are stained with tetramethylrhodamine methyl ester (TMRM). It can bind to
mitochondria and exhibit red fluorescence due to the rhodamine presence in its
structure.
12. Install all three filter cubes. You can find the filter cube holder underneath the
objective lens. Position the DAPI filter cube in the place so that cell nuclei can
be imaged.
13. Move the sample stage to find the cells (cell nuclei as a DAPI filter cube is used)
using either an eyepiece or an image shown in the attached computer. Choose
an appropriate objective (10×, 20×, 30×, etc.) and adjust focus and magnifica-
tion (Fig. 2.18). Save the image (DAPI-stained cell nuclei).
14. Slide the filter cube holder to position the FITC filter cube. Save the image
(FITC-phalloidin-stained actin filaments).
15. Slide the filter cube one more time to position the TRITC filter cube (used for
rhodamine dye). Save the image (TMRM-stained mitochondria).
16. Stack three acquired images into one (Fig. 2.19). Depending on the prepared
slide, staining can vary.
2.12 Laboratory Task 2: Fluorescence Microscopic Imaging of Mammalian Cells 31
Fig. 2.17 A fluorescence

microscope
Fig. 2.18 Cell nuclei are

being focused on the
real-time images shown on
a computer connected to a
microscope
32 2 Cell Culture
Fig. 2.19 Images acquired by a fluorescence microscope. The blue fluorescence image shows
nuclei (top left), the green fluorescence image shows actin filaments (top right), the red fluores-
cence image shows mitochondria (bottom left), and the stacked image is finally shown (bottom
right). (Reprinted with permission from Yoon, 2016. (C) 2016 Springer)
Review Questions
1. Describe three main objectives of cell culture.
2. What is a cell line, and how is it different from a primary cell?
3. What are membrane proteins, and how can they be stable within the cell
membrane?
4. Describe the differences in actin filaments, microtubules, and intermediate filaments.
5. Describe the differences between anchorage-dependent and anchorage-
independent cells.
6. What is focal adhesion? Why is it important for anchorage-dependent cells?
7. What is cell immortalization? How can it be done?
8. What are the negative impacts of cell immortalization?
9. How is a biosafety cabinet different from a chemical hood?
10. Why is CO2 needed for mammalian cell culture?
11. Compare two different incubators for bacterial and mammalian cell culture.
12. Draw a typical cell growth curve. Identify each phase.
Reference
Yoon, J. Y. (2016). Introduction to biosensors (2nd ed.). Springer, Chapter 9. https://doi.

org/10.1007/978-3-319-27413-3_9
Chapter 3
Cell Metabolism
In the previous chapter, we have learned the basics of cell culture. In this chapter,
we will learn the details of cell culture – how we can feed and grow them. Here are
three inquires for you:
Inquiry 1. What is cell metabolism?
How do cells “eat” nutrients and metabolize them with oxygen? And how do
cells generate energy (ATPs) and produce proteins?
Inquiry 2. How do we feed cells?
More specifically, how do we prepare the media for cells? Should we replace or
replenish media periodically? Why do we need to “passage” cells?
Inquiry 3. How can we determine the initial cell number (or density) for tissue engi-
neering applications?
It can be calculated using the first-order growth model under varying cell culture
restrictions.
3.1 What Is Cell Metabolism?
Cells require nutrients (and oxygen) to generate energy, produce proteins, and pro-
liferate (divide) themselves. Nutrients include sugars, amino acids, small molecules,
etc. Energy is generated in the form of ATP (adenosine triphosphate), which is the
primary energy currency in cell metabolism (other forms of energy currency are
also available). Cells can also produce small molecules, such as lactic acid, ammo-
nia, water, etc., as waste or by-products. These processes, taken together, form cell
metabolism. It can be divided into two parts: (1) catabolic reaction, that is, break-
down of complex molecules to generate energy (e.g., ATP) while generating small
molecules as waste or by-product, and (2) anabolic reaction, that is, biosynthesis of

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“Perhaps not, sir, perhaps not,” replied the Major, pityingly. “Do
you never read the ‘Evening Planet,’ sir, when you are at home?”
I winced. The truth was, that I did take in the ‘Evening Planet,’ and
heedfully perused therein the valuable dicta of its eloquent
proprietor, a celebrated parliamentary and platform orator. And I
had been accustomed to give credence to the confident assurance of
this gentleman, that we were miles behind the Northern States of the
American Union in all that was useful and good, and that we could
not do better than copy so shining a model in all things. I had read
and heard the bold statement, made in defiance of statistics, that
America was floating peacefully on the tide of prosperity into the
haven of universal empire—an empire won by bloodless means, of
course; for what nation, unsaddled with an aristocracy, would dream
of war, while Britain was sinking into decrepitude and decay! All this,
and much more, had I heard and read, and I had believed that
Britannia ought to sit at the feet of her flighty offspring for
instruction, and to remodel her old institutions after a republican
pattern. But, as not seldom happens, a nearer view of the United
States did not precisely confirm the loud assertions of the
Americanising party in the British press and senate, and I was
gradually losing my ideal admiration for transatlantic liberty and
customs. After the rapid dinner, and the more leisurely supplement
of juleps and brandy-cobblers imbibed in the bar-room of the hotel, I
asked a coloured waiter if my waggon and mules were forthcoming,
as I was desirous of reaching Nauvoo before dark.
“Iss, massa!” answered the negro, and whisked off with his napkin
to inquire after the lingering equipage.
The Major said he was going to Nauvoo too, and begged the favour
of a lift, which I willingly conceded.
The mules and waggon, with their whipcracking teamster, soon
rattled up to the door; my bill was promptly paid, my baggage
transferred to the vehicle; the Major and I climbed into our places,
and we started.
“How comes it, Major,” said I, “that there is no line open to
Nauvoo?”
The Major knocked the ashes off his cigar as he replied, “Wall, I
suppose it wouldn’t pay. Rail to Fort Madison is all right and spry,
because Uncle Sam has property there; but I guess not a dime could
be drawed from Washington treasury to make a line on to Nauvoo.”
“And from Nauvoo, westward through Iowa, say to Nebraska,”
observed I, with affected carelessness; “what should you say to the
prospects of a railroad in that direction?”
My heart throbbed audibly as I spoke, for all my feigned
indifference, and I listened with anxiety for the Major’s reply. I had
not long to wait.
“That depends,” said my fellow-traveller, with sagacious
deliberation, “on the sort of rail you talk about. Is it a line to go no
farther than Wall Street, and perhaps your London Capel Court, that
you are speaking of, mister?”
“Wall Street and Capel Court! Upon my life, I hardly comprehend
you,” returned I.
“Moonshine, flummery, make-believe, sleepers, rails, stations, all
of paper, that’s what I mean, stranger;” rejoined the Major,
somewhat impatiently.
“But I spoke of a bona fide concern—of a real railway, honestly
made and fairly worked,” answered I; “what would you say to that?”
“Say!” replied the Major, with infinite contempt, “say! Let me see
the gonies. Trot ’em up to me, sir. Just let me have a look at the
simple ones that are at the head of the business, and I’ll tell them
what I think, fast enough. No, Nauvoo is a rising place, a neat
location, but it can wait for a rail one while, unless every sage plant
on the prairie turns to silver dollars.”
After this I asked the Major no more questions. We reached
Nauvoo, and through the dusk I espied the shingled roofs of its
houses, the bold bluffs of limestone, the rushing coffee-coloured
river, and the unfinished building-lots with their heaps of wreck and
rubbish. We put up at the General Jackson Hotel. I had a letter of
introduction to Squire Park of Nauvoo, a gentleman in the flatboat
interest, who owed his title of Squire to his being in the commission
of the peace. But on repairing to his house I was doomed to
disappointment—the more vexatious because Mr Park had been
eulogised by Judge Tips as a man who knew the West thoroughly.
Squire Park was gone to Cairo on business, and was not expected
back before the end of the month. On consulting the map I carried, I
found that a place called Keosauque was the nearest of the few towns
in Iowa to the line of railway, real or imaginary, in connection with
which my name, and those of other men of respectability and
substance, were flaming, in advertisements and on the broadsheets
of a prospectus, throughout the British metropolis. I set off to
Keosauque, mounted on an Indian pony, and accompanied by a
guide in the shape of a wiry backwoodsman, in an enduring costume
of leather, and who gave accommodation to my portmanteau behind
his saddle. For some miles we rode in silence over the apparently
boundless sea of grass, mottled with weeds and flowers, and
occasionally studded with lone farmhouses and maize fields, or by
herds of grazing cattle. Those half-reclaimed mustangs are not the
most pleasant mount for a timid rider, nor am I, George Bulkeley of
Stamford Hill, a very adventurous horseman; and before we had got
far, I began to wish the brute I rode would desist from what seemed
an alternation of starts and stumbles. My guide, a good-humoured
wild man, observed my embarrassment, and undertook its removal.
“See here, Colonel,” said he—strangers in the West are usually
decorated with visionary epaulettes—“you mustn’t keep the rein so
slack as that, nor yet hold your hand up level with your cravat, or,
scalp me, but you’ll be spilt! Mustangs want a tight grip on the bit. So
—steady now. Stick in your knees, Colonel, and scorn to ketch hold of
the pummel—so. Do as you see me do; give him a touch of the spur,
but mind his kicking—for mustangs can kick, they can. You’ll do
nicely, now.”
Ichabod was a skilful riding-master, by instinct, I suppose; and,
thanks to his forcible instructions, I was soon on better terms with
my refractory quadruped. On we rode, over the waving grass,
through the rank weeds, through the belts of cottonwood timber and
maples that skirted every streamlet, and past the swampy bottoms
where sluggish waters wound like wounded snakes. We dined on
dried venison, jerked beef, parched corn, and hominy, at a farm
which did duty for an inn, and slept at another house of the same
character. Next day we resumed our route; and as we rode towards
Keosauque, I ventured to ask Ichabod if he had ever heard of the
Great Nauvoo and Nebraska Railway. I had been hitherto averse to
propounding this query; for how could I tell whether the interests of
my informant might conflict with mine?—but with this rough
frontiersman I felt I was safe. He, at least, was no rival speculator—
no shareholder in a competing line—no steamboat proprietor, or lord
of many stage-waggons. But his first answer was not satisfactory. It
was comprised in the one word, “Anan!”
“The Railway”—asked I again—“from Nauvoo to Nebraska: not a
finished thing, of course; but you surely must have seen or heard of
the works—the bridges, the embankments, and the rest of the
preparations?”
Ichabod shook his head. “You’re talking Greek to me, Colonel, and
that air a fact.”
“How is it possible,” cried I, in an agony, “that there can have been
a railway begun in this country, and the settlers unaware of it? Surely
you must be a stranger to this part of the State yourself!”
“You’re wrong there, Colonel,” answered Ichabod; “I’m Illinois
born, but I’m Iowa bred. In this State I was raised; and I don’t
believe there’s a thing happened over the border sin’ I could mount a
horse, be it buffler or deer, loping Indian, runaway nigger, or Yankee
pedlar, without my hearing on’t. Stop” (and he smote his knee with a
palm as hard as iron)—“I’ve got it. You’re talking of Harvey’s Folly.”
And I thought the young backwoodsman would have tumbled off
his horse in the extravagant burst of mirth which this discovery
produced. “Who-whoop!” cried he; “I’ve seen queer sights, but never
did I think to see a stranger come out in a bee-line from the old
country—no offence, Colonel!—to ax about Harvey’s Folly. I’d nigh
forgot that the thing existed at all. Wah! but it beats coon-catching!”
With some trouble I got an explanation. It appeared from the
borderer’s statement that, years ago, a speculative individual of the
name of Harvey had undertaken to construct a railway from
Nebraska to Nauvoo, with a branch linking it to the Central Illinois
line. He had obtained the usual charter and grant of land from the
State, and had actually commenced operations between Keosauque
and New Buda, two little towns not far from the Missouri boundary.
But he had soon desisted from the Sisyphean task, ruined,
disheartened, or disappointed of the aid on which he had somewhat
sanguinely reckoned; and thenceforth no more had been said of the
scheme or the schemer. “But the property,” groaned I, “the works,
surely they must remain?”
“Why,” said Ichabod, meditatively, “I kinder think there’s rails laid
down a bit—yes, for some miles I guess, and they’ll be there still. The
cussed Indians can’t have stampedoed them, like they do the cattle.
There’s a tidy bridge over a creek or two Harvey built, and some
sheds and scantling; and that’s about all.”
“All,” said I, “think again, Ichabod. Surely there must be more
plant than that, and then the rolling stock?”
The frontiersman laughed. “We know more about gunstocks than
rolling stocks, out here on the pararas,” said he; “and I never heard
of plants, onless ’twas hickory or sumach. But I’ve kinder catalogued
the hull fixings for you, Colonel, without ’tis a pile of rusty iron, or a
few waggon-loads of logs—neat bits of oak timber they were,
trimmed and dressed, and shaped mighty like a saddle-tree, that
Harvey left on the ground.”
“The sleepers, I suppose,” returned I; “are they there still?”
“Well, Colonel, mebbe some of ’em are taking a nap there still,”
replied Ichabod, “but parara men often camp thereabouts, hunting,
cattle-tending, or prospecting, and firewood being mortal scarce on
the plains, ’twasn’t to be expected the bhoys wouldn’t make free with
some chips to cook with. I may have had a chop at those logs with my
tomahawk, when I wanted a broil, onst or twice, myself.”
I groaned again. The Great Nauvoo and Nebraska Railway was
evidently as brittle a speculation as Alnaschar’s basket of glass. I
finished the ride to Keosauque in moody reverie. There was no other
guest to share such rugged plenty as the wooden tavern, called by
courtesy the Eagle Hotel, could afford; and as the landlord was
absent, and the landlady busy in the management of her children
and Irish helps, no one talked to me, and I sat sullen and dejected the
whole evening. Next day, tired as I was, I set out again, under
Ichabod’s guidance, to visit what he persisted in naming Harvey’s
Folly. We reached the spot at last. A swampy level, intersected by
runlets of water, and with a good deal of thorny brake, and here and
there a clump of cottonwood poplars diversifying the scene, had been
selected by Mr Harvey for the site of his preliminary operations. Why
he had chosen that wet ground at all, when so much dry prairie lay
beyond, of very tolerable smoothness, it is difficult to conjecture; but
perhaps the more accurate level had tempted him. There were rails,
certainly there were rails, half-hidden by the growth of hemlocks and
rank grass; but on dismounting I discovered that, for lack of proper
metal trams, the rails had been constructed of wood, covered with a
thin slip of iron—not an unusual device in out-of-the-way parts of
America, as I was afterwards told. The fastenings were very defective,
the sleepers loose, and the whole concern had a crazy haphazard
look. Such as they were, these precious rails were continued for
about 5 miles—5 miles out of 350!—and then they terminated in a
mass of ruin and confusion. There were roofless sheds, scantlings
and screens blown down by hurricane gusts, heaps of rusty iron,
broken tools, damaged wheelbarrows, and a shattered truck with
only one wheel left. Also there were a quantity of sleepers of dressed
oak, and the fragments of many more, split by the axe and charred to
coal, as they lay around the blackened spots of burnt turf, where
many a camp-fire had been lit by the frontiersmen. That was all the
valuable property left at the disposal of the directors. The sight
sickened me. “Harvey’s Folly,” muttered I between my teeth, “say
rather Bulkeley’s Folly—Bulkeley’s credulity, idiocy, weakness! And
not only mine, but Tom Harris’s, and that of all of us. What a long-
eared pack were we to be lured by the crafty piping of such a
dissembling knave as that glib Colonel!” I rode away, sad and
careworn. Ichabod’s quaint talk was unnoticed. I had another
companion that claimed my undivided attention, and that was Care,
Black Care, which sat crouching behind my saddle. I was haunted by
a ghastly phantom of impending bankruptcy. The London Gazette
spread its ill-omened sheet before me, and in its fatal columns I read,
in flaming characters, “George Bulkeley, of Cannon Street in the City
of London, and Stamford Hill, Middlesex, to surrender at Portugal
Street on Monday the 14th inst. Official Assignee, Mr Wilks!” That it
should have come to this! Ruin, ruin, ruin. Ruin and disgrace to us
all, the duped directors of this wretched swindle. Were we not
responsible for the debts of the undertaking? Was not the paid-up
capital in the treacherous hands of our Yankee cashier, Dr Titus A. C.
Bett, and could there be a doubt that it was lost for ever? Plainly the
whole business was a fraudulent trick from the first—a net to catch
gold-fish! Ah! already with my mind’s eye I saw the broker’s men in
possession of Magnolia Villa; I saw my costly furniture, the cellar of
wines I had been so proud of, carriages, pictures, everything,
submitted to public competition by a smirking auctioneer. I heard
the hammer fall, knocking down my Lares and Penates to the highest
bidder. Going, going, gone! the accursed formula rang in my ears
with baleful clearness. Magnolia Cottage to let! My family hiding in
poor lodgings in Boulogne! George Bulkeley, a moody bankrupt,
slinking about the pier of that refuge for insolvency, and afraid to
face the Stock Exchange! Even though the Court might declare me
blameless, even though the commissioner might whitewash me into
commercial purity, my conscience was less complaisant, and sternly
refused me even a third-class certificate.
I might have had the right to ruin myself and family, but what
right had I to make desolate the hearths of many helpless and
confiding people? How about those shareholders ignorant of
business, those pinched vicars, needy widows, poor old half-pay
officers, and the rest, who had been dazzled by our prospectus, and
had invested their savings in the pocket of Dr Titus A. C. Bett? It was
my respectable name, in common with those of my fellows in the
Direction, which had baited the hook for such poor prey as these. My
heart—even City men have hearts sometimes—was heavy and
mournful with a grief not wholly selfish. Plump! fluff! down went the
mustang on his knees, his feet having plunged into the holes that led
to the dwellings of some “prairie-dogs”—interesting little brutes that
burrow all over the plains—and over the animal’s head I flew with the
force of a sky-rocket. Lighting with a great thump on the hard turf, I
ran no trifling risk of a broken neck; but my hat saved me, at the
expense of its own demolition, and I was only stunned. But when
Ichabod hurried to the rescue he found me bruised and faint, and
with a sprained thumb that caused me exquisite pain for the time. So
stupified was I by the shock, that I did not hear the beat of hoofs
upon the green carpet of the prairie, nor the sound of friendly voices,
and was surprised, on looking up, to see that I was surrounded by a
large party of equestrians, who were surveying me from the saddle
with every appearance of interest. Riding-habits and side-saddles
here in prairie-land! hats and feathers, too, of most ladylike elegance,
and a pair of pretty, rather pale faces under the shadow of those
plumed felts. Besides the two girls, there were a grey-haired elderly
man, two younger gentlemen, and three or four mounted blacks in
suits of striped cotton, one of whom led a couple of hounds in a long
leash, while another had a buck strapped behind him on the horse.
“Is the poor gentleman much hurt?” asked one of the young ladies
in a sweet kind voice. Ichabod, as bold as a lion in general, was
awkward and bashful when addressed by a lady, and seemed to be
weighing the words of his answer, when I felt it necessary to reply for
myself. On discovering that I was a stranger in the land, General
Warfield insisted that I should accompany the party to his house,
just across the Missouri border, where my injured thumb should
receive every attention, and where he and his family would gladly
welcome me. Yielding willingly to this hospitable persuasion, I
permitted Ichabod and one of the negroes to help me to remount my
mustang, and we rode towards the Missouri boundary. The family
whose acquaintance I had just made in so singular a way, bore no
similarity to the travelling Americans whom it had previously fallen
to my lot to encounter. General Warfield, his son, daughters, and
nephew, had the well-bred air and unobtrusive demeanour which I
had hitherto deemed exclusively insular. They asked me no abrupt
questions as to my station or errand: they indulged in no diatribes
against my country, nor in any extravagant laudations of their own;
and I might have fancied myself the guest of some long-descended
family at home, but for the wild scenes and unusual objects that met
my eye as we rode along. It turned out that General Warfield, a
retired military officer, not a militiaman, was of an old Virginian
family, and had migrated to the newer soil of Missouri six years ago.
There his children had grown to be men and women, in the hardy
habits of that wild country, a mere outpost of civilisation; and indeed
they were returning from a hunting expedition into Iowa when they
stumbled upon me in my prostrate condition. Three hours’ ride
brought us to the General’s house, a large building of mingled wood
and stone, with a pretty garden on one hand, and on the other the
farm-buildings, the corrals for horses and cattle, and the negro huts.
Within I found furniture of old-fashioned dark mahogany, partridge-
wood, and bird’s-eye maple, old family pictures, pretty knickknacks
picked up during a three years’ residence in Europe, and the massive
silver plate which had been handed down from father to son ever
since the ancestral Warfield settled in Virginia in the reign of Charles
I. I never knew anything so un-American, in respect to the usual
standard of comparison, as the mode of life, the bearing, and tastes,
of General Warfield and his high-spirited and amiable children. Here
was no exaggeration of sentiment, no outrageous national vanity, no
rude indifference to the feelings of others, no prying, no pretension. I
felt, as I conversed with them, how wide was the gulf that severed the
North from the South. It was not diversity of interest alone, but
diversity of habits, principles, and aspirations. Wide apart in heart
and mind as the poles from each other, the citizens of the opposite
ends of the Union had but the feeble Federal bond to delay that
violent disruption and severance of which, even then, the signs of the
times gave fearful warning. But it is not my purpose to linger on the
happy days I spent beneath the roof of my kind hosts. Let me rather
relate the information I received from General Warfield, when his
friendly hospitality had caused me to confide to his ear my errand to
America, and the ruin I had too much reason to anticipate.
“My dear sir,” said the General, “I am glad you have told me of this
—very glad. I can help you in this matter.”
The General then proceeded to tell me that, in the first year of his
residence in Missouri, Harvey, a notorious speculator, had begun the
railway whose miserable wreck I had visited. He had given it up for
want of funds, had become insolvent, and was reputed to have died
in Texas. That he had received a real concession of land and
authentic charters from the State legislatures, was undoubted. But
the concession had been clogged by the express stipulation, that in
two years Harvey should have a hundred and fifty miles in working
order, and that the whole should be completed in four years. The
condition not having been complied with, the concession was null
and void. The Great Nauvoo and Nebraska Railway Company, had no
right to a corporate existence.
“But,” said I, “I of course perused the papers. I saw no mention of
such a conditional clause.”
The General smiled.
“Depend upon it, Mr Bulkeley,” said he, “that erasure and forgery
have been practised to make the old deeds sufficiently tempting to
effect the only purpose their present holders have in view—that of
raising cash in the London market. Colonel Sling—who, by the way,
is no more a colonel, even of militia, than black Cæsar there—is no
novice at fraud. He was convicted at Jefferson city of a like offence,
and I was present at his trial, and heard some of his antecedents;
indeed, I was a witness in the case. But if you will take my advice, you
will hasten back to England, and, if possible, save the funds in the
hands of this confederate of his, this Bett, before the pair can
abscond with their gains. Do not parley, but apply to the police at
once, if, indeed, it be not too late.”
Finally, General Warfield was so good as to accompany me to the
chief town of Iowa State, where he introduced me to the legal
authorities, by whom his statements were fully confirmed, and the
Nauvoo and Nebraska declared a transparent swindle. In this town
we suddenly came on “Colonel” Sling, who had come out by the next
packet, and was tracking me, no doubt in the hope of hoodwinking or
silencing me in some mode or other. But when he saw the General,
his swaggering air collapsed, a guilty crimson suffused his yellow
cheeks, and he slunk away and entered a tavern without accosting us.
And yet when, after giving hearty thanks to my kindly Virginian
friend, I hurried to embark at New York, I had the honour of finding
Colonel Coriolanus Sling, my fellow-passenger. He now ventured to
address me, but by this time I was on my guard against his specious
eloquence, and he retired with an air of mingled effrontery and
shame. At Liverpool, as I took my seat in the train, which I did
without the loss of a moment, I saw Colonel Sling dart into the
telegraph office. So busy was my brain with what was before me, that
I did not, during the principal part of the journey, attach any
particular meaning to this proceeding of my treacherous ally. When I
did think of its probable object, I struck my forehead, and could have
cursed my blind stupidity, my dulness of conception. After all my
haste, scampering as quickly as possible to the station at Liverpool,
was I to be too late, after all? Was this Yankee rascal to be permitted
to warn his brother knave in London through my inattention, and
was the paid-up capital to fatten the two harpies whose tools we had
been? Heavy misgivings filled my heart as I arrived in London,
hurried to Scotland Yard, and requested that a detective policeman
might at once be ordered to accompany me to the residence of Dr
Titus A. C. Bett, cashier to the Nauvoo and Nebraska Company.
Luckily I was a man of credit and character in the city; my request
was granted instantly, and off whirled the hansom cab, as fast as
hansom cab could be impelled by the most lavish bribe, on its way to
Piccadilly, bearing me and a quiet man with a resolute, thoughtful
face, in plain clothes. Ha! there is a cab waiting at the door as we
jump out—I hot and breathless, the policeman cool and steady. The
gaping servant-girl belonging to the lodgings comes quickly at our
knock. It is morning yet, early morning, from a London point of view
—not much after nine.
“Is Dr Bett in?”
“Yes, sir,” replies the girl, “but he’s just a-going. He sent me out for
the cab five minutes ago, and he’s called away so sudden he won’t
take breakfast.”
“Ah, indeed!” says the detective; “telegram, I suppose, eh?”
“Yes, sir,” replied the maid, “and he swore hawful because I hadn’t
woke him up directly it came, two hour ago, along with the milk, but
I didn’t dare, ’cause he always stops out late, and always swears and
scolds if I bring up his hot water before nine o’clock.”
I could have hugged that maid, Mary Ann, Eliza, or Susan, no
matter what, for she was my preserver—a most valuable but
unwitting ally. I did give her a sovereign as I bade her show us up.
We found the Doctor, unshaved, half dressed, tugging at his boots,
and with a leather dressing-case weighty with gold and notes lying on
the table at his elbow. We rushed in with scant ceremony. The
detective tapped him on the shoulder and took him into custody with
the magic formula of uttering her Majesty’s name. The bubble burst,
but the funds were saved; and after some expense, ridicule, and
trouble, we were able to return their money to the shareholders, and
I washed my hands most gladly of my American investment.
THE LANDSCAPE OF ANCIENT ITALY, AS
DELINEATED IN THE POMPEIAN
PAINTINGS.
“Und aber nach zweitausend Jahren

Kam ich desselbigen Wegs gefahren.”
“Et puis nous irons voir, car décadence et deuil

Viennent toujours après la puissance et l’orgueil,
Nous irons voir....”
We are so much accustomed to depend on the four great literary

languages for the whole body of our information and amusement,
that it occurs to few to consider that ignorance of other European
dialects involves any inconvenience at all, except to those who have
occasion to visit the countries in which they are spoken. Yet there is
much of really valuable matter which sees the light only in the minor
tongues, especially those of the industrious North, and with which
the world has never been made familiar through translation.
Joachim Frederic Schouw, the Danish botanist, is one of the writers
of our day who has suffered most prejudicially both to his own fame
and to the public from having employed only his native language. For
his writings are not only valuable in a scientific point of view, but
belong to the most popular order of scientific writing, and would
assuredly have been general favourites, had not the bulk of them
remained untranslated. His ‘Tableau du Climat de l’Italie’ has,
however, appeared in French, and is a standard work. A little
collection of very brief and popular essays, entitled ‘The Earth,
Plants, and Man,’ has been translated both into German and English.
One of these, styled ‘The Plants of Pompeii,’ is founded on a rather
novel idea. The paintings on the walls of the disinterred houses of
that city contain (among other things) many landscape
compositions. Sometimes these are accessory to historical
representations. But they often merely portray the scenery of
ordinary out-door life. The old decorators of the Pompeian chambers
had indeed an evident taste for those trivial tricks of theatrical
deception, which are still very popular in Italy. Their verdure, sky,
and so forth, seem often as if meant to impose on the spectator for a
moment as realities; and are, therefore, executed in a “realistic”
though sketchy style. “Consequently,” says Schouw, “the observation
of the plants which are represented in these paintings will give, as far
as they go, the measure of those which were familiar to the ancient
eye, and will help to show the identities and the differences between
the vegetation of the Campanian plains a hundred years after Christ,
and that which adorns them now.”
We propose to follow the Professor through this confined but
elegant little chapter of his investigations. But by restraining
ourselves to this alone, we should be dealing with only part of a
subject. In most regions, two thousand years have made considerable
changes in the appearance of the vegetable covering of the earth; but
in that land of volcanic influences in which Pompeii stood, great
revolutions have taken place, during that time, in the structure of the
ground itself. Sea and land have changed places; mountains have
risen and sunk; the very outlines and main landmarks of the scene
are other than what they were. Let us for a moment imagine
ourselves gazing with Emperor Tiberius from his “specular height”
on precipitous Capri, at that unequalled panorama of sea and land
formed by the Gulf of Naples, as thence descried, and note in what
respects the visible face of things has changed since he beheld it.
The central object in his view, as in that of the modern observer,
was Vesuvius, standing out a huge insulated mountain mass,
unconformable with the other outlines of the landscape, and covered
then, as now, with its broad mantle of dusky green. Then, as now, its
volcanic soil was devoted to the cultivation of the vine. But in other
respects its appearance was widely different. No slender, menacing
column of smoke rose perpetually from its summit. Nor was it lurid,
at night, with that red gleam of the slow river of fire,
“A cui riluce
Di Capri la marina
E di Napoli il porto e Mergellina.”
It was an extinct volcano, and had been so for unknown ages. Nor
did it exhibit its present characteristic cone, nor probably its double
top; Vesuvius and Somma were most likely one; and the deep half-
moon-shaped ravine of the Atrio del Cavallo, which now divides
them, is thought to be a relic of the ancient crater. That crater was a
huge amphitheatrical depression, several miles in circuit, filled with
pasture-lands and tangled woods. Spartacus and his servile army had
used it not long before as a natural fortress. But this feature was
scarcely visible to the spectator at Capri, opposite the mountain, to
whom the summit must have appeared as a broad flat-topped ridge,
in shape and height very similar to the Table Mountain at the Cape of
Good Hope.
At the time in question, scarcely a few vague traditions remained
to record the fact that the mountain had once “burnt.” The fiery
legends of Magna Græcia related to the country west of Naples,
where volcanic action had been more recent: the Phlegræan fields,
the Market-place of Vulcan (Solfatara), the cone of Gnarime (Ischia),
through which the imprisoned Typhœus breathed flame, from
whence he has been since transferred to Vesuvius, as a Genoese
monk informed us when we and he first looked on that volcano
together. Vesuvius awoke from his sleep of unknown length, as every
one knows, in A.D. 79, when he celebrated his resumption of authority
by that grand “extra night” of the 24th August, which has had no
rival since, in the way of pyrotechnical entertainment, except on the
distant shores of Iceland, the West Indies, and the Moluccas. His
period of activity lasted nearly a thousand years. Then he relapsed
into lethargy for six hundred. In 1631, he had resumed (as old prints
show), something nearly resembling the form which we have
attributed to him in classical times. His top, of great height, swollen
up by the slow accumulation of burning matter, without a vent, was a
level plateau, with a pit-like crater, filled with a forest of secular oaks
and ilexes: only a few “fumaroles,” or smoke-holes, remained here
and there to attest his real character. Even the legends of his
conflagrations had become out of date. The old “Orearch” or
mountain-spirit, Vesevus, is portrayed by the local poet Pontanus in
the fifteenth century, as a rustic figure, with a bald head, hump back,
and cincture of brushwood—all fiery attributes omitted. Even his
terrible name was only known to the learned: the people called him
the “Monte di Somma.” The suburban features of a great luxurious
city, convents, gardens, vineyards, hunting-grounds, and parks of the
nobility, had crept again up the sides of the mountain, until they
almost mingled with the trees on the summit. The approaching hour
was not without its premonitory signs, many and strange. The
phenomena which Bulwer makes his witch of Vesuvius recount, by
way of warning, to Arbaces, are very closely borrowed from
contemporary narratives of the eruption of 1631. Nor were the omens
of superstition wanting, accommodated to the altered feelings of the
times. At the Plinian eruption, the people imagined that the old
giants buried in the Phlegræan fields had risen again, and renewed
their battle with the gods: “for many phantoms of them,” says Dio
Cassius, “were seen in the smoke, and a blast, as of trumpets, was
heard.” In 1631, carriages full of devils were seen to drive, and
battalions of diabolical soldiers to gather in marching array along the
precipitous flanks of the mountain. The footsteps of unearthly
animals were tracked on the roads. “A peasant of the name of
Giovanni Camillo” (so we are informed by the Jesuit Giulio Cesare
Recupito, a contemporary), “had passed Easter Eve at a farm-house
of his own on the mountain. There, without having taken a mouthful
of anything, he was overtaken by a profound slumber, from which
awakening suddenly, he saw no longer before his eyes the likeness of
the place where he had fallen asleep, but a new heaven, a new soil, a
new landscape: instead of a hill-side covered with wood, there
appeared a wall crossing the road, and extending on each side for a
great distance, with a very lofty gate. Astonished at this new scene,
he went to the gate to inquire where he was. There he found a porter
of the order of St Francis, a young man in appearance. Many
conjecture that this was St Antony of Padua. The porter at first
seemed to repulse him, but afterwards admitted him into the
courtyard, and guided him about. After a long circuit they arrived at
a great range of buildings breathing fire from every window.” In
short, the poor peasant was conducted, after the fashion of such
visions, through the mansions of hell and purgatory, where he saw,
of course, many of his acquaintance variously tormented. “At last, on
the following day, he was restored to himself, and to Vesuvius: and
was ordered to inform his countrymen that a great ruin was
impending over them from that mountain: wherefore they should
address their vows and prayers to God. On Easter Day, at noon, he
came home, and was observed of many with his dress sprinkled with
ashes, his face burnt black, as if escaped from a fire.” This was two
years before the eruption, and during the interval Camillo always
told the same story; wherefore, after passing a long time for either
mad or drunk, he was finally raised to the dignity of a prophet. At
last, on the night of the 15th December, the ancient volcano
signalised his awakening by a feat of unrivalled grandeur. In forty-
eight hours of terrific struggles, he blew away the whole cap of the
mountain; so that, on the morning of the 18th, when the smoke at
last subsided, the Neapolitans beheld their familiar summit a
thousand feet lower than it had been before; while its southern face
was seamed by seven distinct rivers of fire, slowly rolling at several
points into the sea.
Since 1631, the frequency, if not the violence, of the eruptions
seems to have gradually increased, and Vesuvius is probably more
“active” now, in local language, than at any former time in his annals,
having made the fortunes of an infinity of guides and miscellaneous
waiters on Providence within the last twelve years, besides burning a
forest or two, and expelling the peasantry of some villages. But his
performances on a grand scale seem for the present suspended.
Frequent eruptions prevent that accumulation of matter which
produces great ones. Indeed, the late Mr Laing, whose ‘Notes of a
Traveller’ show him to have been that identical “sturdy Scotch
Presbyterian whig” who visited Oxford in company with Lockhart’s
Reginald Dalton, “reviling all things, despising all things, and puffing
himself up with all things,” deliberately pronounced the volcano a
humbug, and believed the depth of its subterranean magazines to be
extremely trifling. Still, the curious traveller, like that fabulous
Englishman who visited the lion-tamer every night for the chance of
seeing him devoured, cannot help looking with a certain eagerness
for the occurrence of those two interesting catastrophes, of which the
day and hour are written down in the book of the Fates—that
combination of high tide, west wind, and land-flood, which is to
drown St Petersburg; that combination of south-east wind and first-
class eruption which is to bury Naples in ashes. This finale seemed
nearer in that recent eruption of December 1860, which spent its
fury on Torre del Greco, than perhaps on any former occasion; but
once more the danger passed away.
To return, however, from this digression, which has nothing to
excuse it except the interest which clings even to often-repeated
stories respecting the popular old volcano. Other features in that
wonderful panorama, seen from Capri, have undergone scarcely
inferior changes since the time of Tiberius. Yonder rich tract of level
land at the mouth of the Sarno, between Torre dell’ Annunziata and
Castellamare, did not exist. The sea has retreated from it. Tiberius
saw, instead of it, a deep bay washing the walls of the compact little
provincial city of Pompeii. But the neighbouring port of Stabiæ is
gone: not a vestige of its site remains. Above it to the right, Monte
Sant’Angelo, and the limestone sierra of which it forms a part,
remain, no doubt, unchanged by time. Only that marvellous range of
Roman villas and gardens which lined its foot for leagues, almost
rivalling the structures of the opposite Bay of Baiæ for magnificence,
has disappeared, no one knows how or when. The diver off the coast
of Sorento can touch with his hand the long ranges of foundation-
work, brick and marble, which now lie many feet beneath the deep
clear water. It was a strange fit of short-lived magnificence, that
which induced the grandest of millionnaires, the chiefs of the
Augustan age, to raise their palaces, all round the Gulf of Naples, on
vaulted ranges of piles laid within the sea, so that its luxurious ripple
should be heard under the rooms in which they lived. Niebuhr, who,
with all his curious insight into the ways of antiquity, was not
superior to the temptation of finding a new reason for everything,
asserts that they did so in order to escape the malaria. But that
mysterious evil influence extended some way beyond the shore. The
country craft will, to this day, keep as far as they can, in the summer
nights, off the coast of the Campagna, while the quiet land-breeze is
wafting death from the interior. The real causes were, doubtless,
what the writers of the time disclose. The land close to the shore was
dear and scanty, and ill-accommodated for building, from its
steepness. The first new-comer who set the fashion of turning sea
into land, was imitated by others in the mere wantonness of wealth,
until the whole shore became lined with palatial edifices, like the
Grand Canal of Venice; but not so durably. These classical structures,
frequently delineated with more or less detail in the Pompeian
frescoes, were as beautiful and as transitory as those of our dreams;
or like the vision which Claude Lorraine transferred to canvass in the
most poetical of landscapes, his ‘Enchanted Palace.’ Judging from
the singular phenomena exhibited by the ‘Temple of Serapis,’ and by
other topographical records, geologists have concluded that land and
sea, in this volcanic region, wax and wane in long successions of
ages. Thus the sea rose (or rather the land sank) on the coast of the
Bay of Naples for about eleven centuries previous to A.D. 1000; then
the reverse movement took place until about A.D. 1500: and the land
is now sinking again. If so, these marine palaces must have gradually
subsided into the sea, and their owners may have been driven out by
the invasion of cuttle-fish and sea-hedgehogs, and other monsters of
the Mediterranean shallows, in their best bedrooms, even before
Norman or Saracen incursions had reduced them to desolation. But
whatever the cause of their disappearance, they had vanished before
modern history began: nor has modern luxury, in its most profuse
mood, ever sought to reproduce them. Their submarine ruins remain
as memorials of ages when men were at all events more daring and
earnest in their extravagance, and the “lust of the eye and the pride
of life” were deified on a grander scale, than at any other epoch of the
world’s history.
Naples herself, the “idle” and the “learned” (for the ancients called
her somewhat inconsistently by both epithets, nor had she as yet
acquired her more recent soubriquet of the “beautiful”), formed a far
less conspicuous object in the view than now; it was a place of some
twenty or thirty thousand souls, according to Niebuhr’s conjectural
estimate; confined between the modern Mole on the one hand, and
the Gate del Carmine on the other; and nestling close in the
neighbourhood of the sister city Herculaneum. The lofty line of the
houses on the Chiaia—of which you may now almost count the
windows in the top storeys from the sea-level at Capri, through that
pellucid atmosphere, while the lower storeys are hidden by the
earth’s curvature—did not then exist. But instead of it there extended
the endless terraces and colonnades, the cypress avenues and plane
groves, of that range of fortress-palaces erected by Pollio and
Lucullus, enlacing island, and beach, and ridge, even to the point of
Posilippo, with tracery of dazzling marble. Here, however, the mere
natural changes have been small, except that an island or two (like
that of the Castel dell’ Uovo) has since been joined to the continent.
But farther west, round the Bay of Baiæ, fire and water have dealt
most fantastically with the scenery. Scarcely a prominent feature on
which the Roman eye rested remains unchanged. Quiet little Nisida
was a smoking semi-volcano. Yonder level dun-coloured shore, from
Pozzuoli to the Lucrine, was under water, and the waves dashed
against a line of cliff now some miles inland. That crater-shaped Lake
of Agnano, now the common resort of Neapolitan holiday-makers,
did not exist; it must have been formed by some unrecorded
convulsion of the dark ages. Yonder neatly truncated cone, rising five
hundred feet above the plain, seems as permanent a feature in the
landscape as any other of the “everlasting hills;” but it was the
creation of a few days of violent eruption, only three centuries ago—
as its name of Monte Nuovo still indicates—whether by “upheaval” or
by “ejection,” philosophers dispute. But the beautiful Lucrine Lake,
the station of Roman fleets and the very central point of Roman
luxury, disappeared in the same elemental commotion; leaving a
narrow stagnant pool behind. Only yon slight dyke or barrier of
beach, between this shrunken mere and the sea, deserves respect; for
that has remained, strange to say, almost unaltered throughout. It is
one of the very oldest legendary spots of earth; doubtless the very
road along which Hercules dragged the oxen of Geryon; the very
“narrow shore” on which Ulysses landed, in order to call up the
melancholy shades of the dead. Farther inland, again, Avernus
remains unchanged, in shape at least; but many and strange are the
revolutions which it has undergone in other respects. We first hear of
it as a dark pool, surrounded by forests; the bed, doubtless, of an
ancient crater filled with water, and retaining much of volcanic
action; but not (as commonly supposed) fatal to the birds that flew
over it. That notion is not classical; or rather, it is founded on a
misconception of classical authorities. The pool is not called by the
best writers “lacus Avernus” but “lacus Averni,” the lake of the
Avernus. What is an Avernus? Lucretius tells us that it is a spot
where noxious gases escape from the earth, so that the birds which
fly over it fall dead on the earth or into the lake if there happens to be
a lake below them.
“Si forte lacus substratus Averno est.”
And Virgil’s description, accurately construed, gives exactly the

same meaning.
“Spelunca alta fuit....
....tuta lacu nigro nemorum que tenebris,
Quam super” (not quem super, over the cavern, not the lake)
....“haud ullæ poterant impune volantes
Tendere iter pennis....
Unde locum” (not lacum) “Graii dixerunt nomine Aornon.”
It was the exhalations from the mysterious cavern that were

deadly, not those from the lake. Such an “Avernus” is the “Gueva
Upas” or Valley of Death, in Java, to which condemned criminals
were formerly sent to perish; whence the romance about the Upas
Tree. And such an Avernus, on a small scale, still exists on the shore
of the peaceful little Lake of Laach in Germany, also an extinct
crater: there are spots on its beach where bird-corpses are to be
found in numbers, killed by mephitic exhalations. But—to return to
our lake—it must at that time have lain at or (like some other extinct
craters) below the level of the sea; for Augustus’s great engineering
operation consisted in letting the sea into the lake.
“Tyrrhenusque fretis immittitur æstus Avernis.”
Fifteen hundred years afterwards, and just before the Monte

Nuovo eruption, the place was visited by that painful old
topographer, Leandro Alberti, the Leland of Italy. The channel made
by Augustus was then gone; but the lake was still on a level with the
sea, for he asserts that in storms the sea broke into it: and the water,
as he expressly affirms, was salt. Now, its level is several feet above
that of the sea, and the water is fresh. The upheaval must have been
gradual and peaceful, for the outline of the lonely mere is as perfectly
rounded now as the poet Lycophron described it;—but a portion only
of that bewildering succession of changes of which this coast has
been the theatre: the latest vibration of that vast commotion figured
in the legendary war of the Giants. Nor is it quite so wild a conjecture
as some have deemed it, that the tradition which peopled this bright
coast with Cimmerians—then dwellers in the everlasting mist, on the
border-land between the dead and the living—had its origin in the
tales of primeval navigators, who had visited the neighbourhood
during some mighty and prolonged eruption, covering sea and shore
with a permanent darkness which “might be felt:” like the coast of
Iceland in 1783, when for a whole summer continual eruptions arose
from the sea as well as the land: when “the noxious vapours that for
many months infected the air, enveloped the whole island in a dense

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Tissue Engineering: A Primer with

Laboratory Demonstrations 1st Edition

Biochemical Engineering: A Laboratory Manual 1st

Cardiac Tissue Engineering Kareen L.K. Coulombe

Tissue Engineering 3rd Edition Clemens Van Blitterswijk

A Primer on Scientific Programming with Python Texts in

PDE Toolbox Primer for Engineering Applications with

Primary Mathematics 3A Hoerst

Vascular Tissue Engineering Feng Zhao Kam W Leong

Tissue Engineering: Applications and Advancements 1st

ISBN 978-3-030-83695-5 ISBN 978-3-030-83696-2 (eBook)

© Springer Nature Switzerland AG 2022

I have been teaching a semester-long upper-division undergraduate college class on

Tucson, AZ, USA Jeong-Yeol Yoon

3.5 Cell Metabolism: Glutaminolysis and TCA Cycle �������������������������� 39

6.7 Bone Biomaterials, Apatite, and Bioglass���������������������������������������� 107

9.12 Adding Porosity and Nanostructure to 3D Printing�������������������������� 168

12.5 Scaffold for Tissue-Engineered Skin Transplant���������������������������� 223

In this chapter, we will learn several different definitions of tissue engineering as

1.1 Narrow Definition of Tissue Engineering

© Springer Nature Switzerland AG 2022 1

1.2  arly Attempt in Scaffold Development:

1.3 Simple TE Transplant Example: Skin

Fig. 1.5 Bilayered

hydrogel. Hydrogels contain as much as >90% water, which is similar to human

1.4 Simple TE Transplant Example: Pancreas

1.5  xpanded Definition of Tissue Engineering:

Another emerging application of tissue engineering is organ-on-a-chip or simply

Fig. 1.7 Lab-on-a-chip is

1.6 Overview of This Book

create a fully functioning tissue-engineered transplant or tissue/organ mimic (OOC).

Fig. 1.9 Overview of this book

2.1 What Is Cell Culture?

© Springer Nature Switzerland AG 2022 13

Fig. 2.1 HeLa cells with

2.2 Cell Physiology: Cell Membrane and Cytoskeleton

Intermediate filaments are rope-like fibers made from a heterogeneous family of

2.3 Focal Adhesion

Fig. 2.4 Focal adhesion of an anchorage-dependent cell to a surface

a tight adhesion of cells to a surface. Focal adhesion will be further discussed in

2.4 Cell Classification: Anchorage-Dependent Versus

2.5 Cell Classification: Normal Versus Immortalized Cells

Fig. 2.5 Top: Chinese

2.6 Cell Classification: Normal Versus Stem Cells

In the previous section, we briefly discussed differentiation. All cells in a human

2.7 Maintaining Sterile Environment: Biosafety Cabinet

Cell culture can be contaminated by bacteria or yeast, which is quite a common

The core components of a biosafety cabinet are (1) a high-efficiency particulate

2.8 Maintaining Sterile Environment: Autoclave

An autoclave is another essential piece of equipment for maintaining a sterile labo-

Fig. 2.8 Autoclave

2.9 Cell Culture: CO2 Incubator

Cell culture can be conducted in either an incubator or a bioreactor. While a sophis-

2.10 Cell Imaging: Fluorescence Microscope

Fig. 2.10 LB (left) and lyophilized powder of E. coli K12 (right)

blue-­fluorescent dye, actin filaments with a green-fluorescent dye, and mitochon-

2.11 Laboratory Task 1: Bacterial Cell Culture

While mammalian cells are cultured in tissue engineering applications, we will

Objective 3. Spectrophotometric Quantification

Tucson, AZ, USA Jeong-Yeol Yoon

3.5 Cell Metabolism: Glutaminolysis and TCA Cycle �� 39

6.7 Bone Biomaterials, Apatite, and Bioglass�� 107

9.12 Adding Porosity and Nanostructure to 3D Printing�� 168

12.5 Scaffold for Tissue-Engineered Skin Transplant�� 223

1.1 Narrow Definition of Tissue Engineering

1.2 arly Attempt in Scaffold Development:

1.3 Simple TE Transplant Example: Skin

1.4 Simple TE Transplant Example: Pancreas

1.5 xpanded Definition of Tissue Engineering:

1.6 Overview of This Book

2.1 What Is Cell Culture?

2.2 Cell Physiology: Cell Membrane and Cytoskeleton

2.3 Focal Adhesion

2.4 Cell Classification: Anchorage-Dependent Versus

2.5 Cell Classification: Normal Versus Immortalized Cells

2.6 Cell Classification: Normal Versus Stem Cells

2.7 Maintaining Sterile Environment: Biosafety Cabinet

2.8 Maintaining Sterile Environment: Autoclave

2.9 Cell Culture: CO2 Incubator

2.10 Cell Imaging: Fluorescence Microscope

blue-fluorescent dye, actin filaments with a green-fluorescent dye, and mitochon-

2.11 Laboratory Task 1: Bacterial Cell Culture

2.12 aboratory Task 2: Fluorescence Microscopic Imaging

3.1 What Is Cell Metabolism?