Table Of ContentMethods in
Molecular Biology 1817
Mario Baratta Editor
Epithelial
Cell Culture
Methods and Protocols
M M B
ethods in olecular iology
Series Editor:
John M. Walker
School of Life and Medical Sciences
University of Hertfordshire
Hatfield, Hertfordshire, AL10 9AB, UK
For further volumes:
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Epithelial Cell Culture
Methods and Protocols
Edited by
Mario Baratta
Department of Veterinary Science, University of Turin, Turin, Italy
Editor
Mario Baratta
Department of Veterinary Science
University of Turin
Turin, Italy
ISSN 1064-3745 ISSN 1940-6029 (electronic)
Methods in Molecular Biology
ISBN 978-1-4939-8599-9 ISBN 978-1-4939-8600-2 (eBook)
https://doi.org/10.1007/978-1-4939-8600-2
Library of Congress Control Number: 2018946598
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About the Editor
Mario Baratta is full professor of veterinary physiology at the University of Turin (IT). He
received his master’s degree in veterinary medicine at the University of Parma (IT) and his
PhD in neuroendocrinology of animal farm at the University of Bologna (IT). Today he is
the coordinator of the Graduate School program in Veterinary Science for Animal Health
and Food Safety, University of Turin. His main interests are the biology of adult stem cells
in different tissues, and he has developed specific knowledge in functional assessment sys-
tems of epithelial cells and cell differentiation in mammary gland and in muscle in animal
science.
v
Preface
Epithelial tissues line the cavities and surfaces of blood vessels and organs throughout the
body, and epithelial cells exert a fundamental role for absorption or secretion or to act as a
barrier. They are characterized by common structural features, especially their arrangement
into cohesive sheets, but have diverse functions made possible by many specialized adapta-
tions. Many of the physical properties of epithelial cells rely on their attachment to each
other, which is mediated by several types of cell junctions. The specialized functions of
epithelial cells are mediated both through structural modifications of their surface and by
internal modifications, which adapt cells to manufacture and secrete a product. Thus, many
aspects of cellular regulation of different organs are mediated by these cells, and its under-
standing is very important for defining the therapeutic approach in dysregulation. Further,
epithelial cell markers can be used to investigate many aspects of epithelial cell biology
including embryonic development, tissue organization, carcinogenesis, and epithelial-to-
mesenchymal transition status. In the understanding of the cellular physiology of these
particular cell types, the analysis in a three-dimensional environment in which the cellular
polarity is respected is increasingly important.
This book aims to provide a broad overview of the systems and the epithelial cell cul-
ture techniques used in recent years in several animal models. Particular attention was paid
to systems that seek to mimic the three-dimensional organization or a paracrine relation-
ship between the different most physiological cell types. This aspect is considered of par-
ticular importance in the effort to refine the most adequate solutions for the reduction of
in vivo experiments and collect more data compatible with the physiological regulations in
various biological systems.
In particular, the three-dimensional culture permits to investigate deeply these cells since
they are uniquely positioned at the interface where self and non-self meet. In the lung, epi-
thelial cells must separate the airways, and potential harmful materials within them, from the
bloodstream while allowing the free diffusion of oxygen and carbon dioxide. The gastroin-
testinal tract is an even more challenging environment, for in addition to preventing luminal
toxins, microbiota, and microbial products from accessing deeper tissues, the intestinal epi-
thelium must support vectorial, or directional, transport of nutrients, ions, and water.
This book has included the protocols of analysis of epithelial cells from different organs
such as lung (Chapter 4), different parts of gastrointestinal system (Chapters 5, 11–13,
15–18), thyroid (Chapters 1, 2), salivary gland (Chapter 3), ovary (Chapters 8–10), and
mammary gland (Chapters 14, 17). The collection has inserted also some protocols of cul-
tivation of some cell types that from an embryogenic point of view are object of study for
their epithelial derivation as the granulosa cells of which, however, recent studies have pro-
posed a common derivation with the ovarian surface epithelial cells from a precursor cell
called gonadal-ridge epithelial-like cell. Since their applicative importance is undoubted in
the understanding of the reproductive regulations in both human and animal fields, differ-
ent approaches for their manipulation are proposed. For the same reason, a chapter on
amniotic epithelial cell has been included (Chapter 7). A specific chapter has been dedicated
vii
viii Preface
to the olfactory epithelial tissue for the interesting role that this model plays in the study of
neurogenesis (Chapter 19).
Another aspect that the edition would like to propose is the translational aspect of
research that uses these study models. In fact, the study of epithelial cells in vitro attracts
researchers engaged in different scientific areas of life sciences and how these different skills
can integrate and enrich each other.
The contributions of experimental models come from laboratories actively engaged in
research in very recent years. For this reason, the book aims to be a very updated review of
the culture methods applied to epithelial cells in functional studies.
I hope that this edition will be of particular interest among young researchers who are
involved in the study and use of cell culture techniques in epithelial cell physiology where
in vitro techniques are increasingly considered for the replacement of experimental
approaches in vivo.
Turin, Italy Mario Baratta
Contents
About the Editor � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � v
Preface � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � vii
Contributors ���������������������������������������������������������� � xi
1 Normal Human Thyrocytes in Culture. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Sarah J. Morgan, Susanne Neumann, and Marvin C. Gershengorn
2 Isolation and Culture of Juvenile Pig Thyroid Follicular Epithelia. . . . . . . . . . . 9
James D. Lillich and Peying Fong
3 Reassembly of Functional Human Stem/Progenitor Cells in 3D Culture . . . . . 19
Danielle Wu, Patricia Chapela, and Mary C. Farach-Carson
4 Culture and Differentiation of Lung Bronchiolar Epithelial
Cells In Vitro . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
Dahai Zheng and Jianzhu Chen
5 Differentiation of Gastrointestinal Cell Lines by Culture
in Semi-wet Interface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
Macarena P. Quintana-Hayashi and Sara K. Lindén
6 Three-Dimensional Cell Culture Model Utilization in Renal Carcinoma
Cancer Stem Cell Research. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
Kamila Maliszewska-Olejniczak, Klaudia K. Brodaczewska,
Zofia F. Bielecka, and Anna M. Czarnecka
7 Amniotic Epithelial Cell Culture. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67
Angelo Canciello, Luana Greco, Valentina Russo, and Barbara Barboni
8 Bovine Granulosa Cell Culture. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79
Bushra T. Mohammed and F. Xavier Donadeu
9 Bioencapsulation of Oocytes and Granulosa Cells . . . . . . . . . . . . . . . . . . . . . . . 89
Massimo Faustini, Giulio Curone, Maria L. Torre, and Daniele Vigo
10 Ovine Granulosa Cells Isolation and Culture to Improve Oocyte Quality . . . . . 95
Giovanni Giuseppe Leoni and Salvatore Naitana
11 3D Model Replicating the Intestinal Function to Evaluate
Drug Permeability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107
Inês Pereira, Anna Lechanteur, and Bruno Sarmento
12 Isolation of Human Gastric Epithelial Cells from Gastric Surgical
Tissue and Gastric Biopsies for Primary Culture . . . . . . . . . . . . . . . . . . . . . . . . 115
Jinhua Qin and Xuetao Pei
13 Long-Term Culture of Intestinal Organoids . . . . . . . . . . . . . . . . . . . . . . . . . . . 123
Seung Bum Lee, Sung-Hoon Han, and Sunhoo Park
ix
x Contents
14 Bovine Mammary Organoids: A Model to Study Epithelial Mammary Cells . . . 137
Eugenio Martignani, Paolo Accornero, Silvia Miretti, and Mario Baratta
15 Establishment of Human- and Mouse-Derived Gastric Primary
Epithelial Cell Monolayers from Organoids. . . . . . . . . . . . . . . . . . . . . . . . . . . . 145
Emma Teal, Nina Bertaux-Skeirik, Jayati Chakrabarti, Loryn Holokai,
and Yana Zavros
16 Mouse-Derived Gastric Organoid and Immune Cell Co-culture
for the Study of the Tumor Microenvironment. . . . . . . . . . . . . . . . . . . . . . . . . 157
Jayati Chakrabarti, Loryn Holokai, LiJyun Syu, Nina Steele,
Julie Chang, Andrzej Dlugosz, and Yana Zavros
17 Murine and Human Mammary Cancer Cell Lines: Functional Tests . . . . . . . . . 169
Paolo Accornero, Eugenio Martignani, Silvia Miretti, and Mario Baratta
18 In Vitro Porcine Colon Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 185
Matheus O. Costa, Janet E. Hill, Michael K. Dame, and John C. S. Harding
19 Primary Cultures of Olfactory Neurons from the Avian
Olfactory Epithelium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 197
George Gomez
Index. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 209
Contributors
Paolo accornero • Department of Veterinary Science, University of Turin, Grugliasco,
TO, Italy
Mario Baratta • Department of Veterinary Science, University of Turin, Grugliasco, TO,
Italy
BarBara BarBoni • Faculty of Bioscience and Technology for Food, Agriculture and
Environment, University of Teramo, Teramo, Italy
nina Bertaux-Skeirik • Department of Pharmacology and Systems Physiology,
University of Cincinnati, Cincinnati, OH, USA
Zofia f. Bielecka • Department of Oncology with Laboratory of Molecular Oncology,
Military Institute of Medicine, Warsaw, Poland; School of Molecular Medicine,
Warsaw Medical University, Warsaw, Poland
klaudia k. BrodacZewSka • Department of Oncology with Laboratory of Molecular
Oncology, Military Institute of Medicine, Warsaw, Poland
angelo canciello • Faculty of Bioscience and Technology for Food, Agriculture and
Environment, University of Teramo, Teramo, Italy
Jayati chakraBarti • Department of Pharmacology and Systems Physiology,
University of Cincinnati, Cincinnati, OH, USA
Julie chang • Department of Biomedical Engineering, University of Cincinnati,
Cincinnati, OH, USA
Patricia chaPela • Department of Diagnostic and Biomedical Sciences, School of
Dentistry, University of Texas Health Science Center at Houston, Houston, TX, USA;
Department of BioSciences, Rice University, Houston, TX, USA
JianZhu chen • Interdisciplinary Research Group in Infectious Diseases,
Singapore- Massachusetts Institute of Technology Alliance for Research and Technology,
Singapore, Singapore; The Koch Institute for Integrative Cancer Research and
Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, USA
MatheuS o. coSta • Department of Large Animal Clinical Sciences, Western College of
Veterinary Medicine, University of Saskatchewan, Saskatoon, SK, Canada; Department
of Farm Animal Health, Faculty of Veterinary Medicine, Utrecht University, Utrecht,
The Netherlands
giulio curone • Dipartimento di Medicina Veterinaria, Università degli Studi di
Milano, Milan, Italy
anna M. cZarnecka • Department of Oncology with Laboratory of Molecular Oncology,
Military Institute of Medicine, Warsaw, Poland
Michael k. daMe • Division of Gastroenterology, Department of Internal Medicine,
University of Michigan Medical School, University of Michigan, Ann Arbor, MI, USA
andrZeJ dlugoSZ • Department of Dermatology, University of Michigan, Ann Arbor,
MI, USA; Department of Cell and Developmental Biology, University of Michigan,
Ann Arbor, MI, USA
xi
