Mouse Cell Culture: Methods and Protocols

262 Pages·2010·8.39 MB·English

by  Melany Jackson, A. Helen Taylor, Andrew Ward

TM
METHODS IN MOLECULAR BIOLOGY
Series Editor
John M. Walker
School of Life Sciences
University of Hertfordshire
Hatfield, Hertfordshire, AL10 9AB, UK
For other titles published in this series, go to
www.springer.com/series/7651
Mouse Cell Culture
Methods and Protocols
Edited by
AndrewWard
University of Bath, Bath, UK
David Tosh
University of Bath, Bath, UK
Editors
David Tosh
Andrew Ward
Department of Biology &
Department of Biology &
Biochemistry
Biochemistry
University of Bath
University of Bath
Claverton Down
Claverton Down
Bath
Bath United Kingdom BA2 7AY
United Kingdom BA2 7AY
Preface
Techniques for the isolation, maintenance and growth of tissues normally found in a mul-
ticellular organism can be traced back for over 100 years. The pioneering developmental
biologist Wilhelm Roux is generally attributed with the first reported tissue culture exper-
iment after maintaining an explant of chick medullary plate for several days in a warm
saline solution. This experiment recognised the need to provide cultured cells with condi-
tions that resemble their normal environment, including an optimal temperature and an
isotonic medium, and could be considered the founding principle of tissue culture. Tissue
culture has become more sophisticated since then mainly through the ability to increas-
ingly recognise the conditions needed to support specific cell types and, indeed, to instruct
their behaviour in vitro. Tissue culture experiments have been used to address many dif-
ferent biological questions. Most obviously, cultured cells have been used to study the
properties of the tissues from which they are derived. The accessibility of cultured cells,
combined with the ability to expand a homogeneous cell population from a relatively lim-
ited source, opens up a wealth of possibilities for researchers. Cultured cells have been
used to manufacture protein products, and, as a test bed for new drugs, they have been
developed to model features of human disorders and, conversely, as a source of tissue
replacement in human disease, providing new possibilities for regenerative medicine.
The mouse is the genetic model of choice for those interested in understanding mam-
malian growth, development, behaviour and physiology. Methods for manipulation of the
mouse genome have rapidly advanced and become widespread in their application. They
offer a powerful means to investigate gene function; however, in order to understand the
mechanisms that underlie the phenotypes of transgenic and knockout mice, the applica-
tion of a whole range of additional techniques is required. Cell culture describes a set
of techniques that have been invaluable in its own right and can be seen as increasingly
important when used in conjunction with the resources generated by mouse genetic exper-
iments. This book brings together a number of methods for the culture of specific cells
and tissues isolated from the foetal or adult mouse. Methods have been developed for the
culture of a wide range of cell types, and this range is still steadily expanding. Indeed, for
any tissue or cell type, culture methods continue to evolve. Thus, although we could not
hope to be comprehensive in the coverage of different tissues, our main aim in compil-
ing this book was to bring together a selection of the current methods in order to make
them available in one convenient source. We have included protocols for the explant of
foetal tissues and stem cells that allow developmental processes to be followed ex vivo, as
well as protocols for the culture of isolated cell types that allow for the study of relatively
homogeneous cell populations. The result is a diverse collection of protocols that cover a
number of intensively studied systems using a variety of techniques. These methods should
not only be immediately applicable by many researchers but also be useful as a springboard
for the development of new tissue culture methods.
Andrew Ward
David Tosh
v
Contents
Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix
1. The Culture of Mouse Embryonic Stem Cells and Formation
of Embryoid Bodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Melany Jackson, A. Helen Taylor, Elizabeth A. Jones,
and Lesley M. Forrester
2. Derivation of Primary Mouse Embryonic Fibroblast (PMEF) Cultures . . . . . . 19
Alastair S. Garfield
3. Embryonic Skeletal Muscle Microexplant Culture and Isolation
of Skeletal Muscle Stem Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
Janet Smith and Deborah Merrick
4. The Embryonic Kidney: Isolation, Organ Culture, Immunostaining
and RNA Interference . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57
Jamie A. Davies
5. Explant Culture of Mouse Embryonic Whole Lung, Isolated Epithelium,
or Mesenchyme Under Chemically Defined Conditions as a System
to Evaluate the Molecular Mechanism of Branching Morphogenesis
and Cellular Differentiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
Pierre-Marie del Moral and David Warburton
6. Isolation, Culture, and Characterisation of Mouse Embryonic
Oesophagus and Intestine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81
Jonathan M. Quinlan, Wei-Yuan Yu, and David Tosh
7. Isolation and Culture of Embryonic Pancreas and Liver . . . . . . . . . . . . . 91
Zoë D. Burke, Wan-Chun Li, Jonathan M.W. Slack, and David Tosh
8. Isolation and Culture of Mouse Satellite Cells . . . . . . . . . . . . . . . . . . 101
Antonio Musarò and Laura Barberi
9. Analysis of Cardiac Myocyte Biology in Transgenic Mice: A Protocol
for Preparation of Neonatal Mouse Cardiac Myocyte Cultures . . . . . . . . . . 113
Nigel J. Brand, Enrique Lara-Pezzi, Nadia Rosenthal,
and Paul J.R. Barton
10. Short- and Long-Term Cultivation of Embryonic and Neonatal Murine
Keratinocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125
Reto Caldelari and Eliane J. Müller
11. Isolation, Culture and Analysis of Mouse Mammary Epithelial Cells . . . . . . . 139
Matthew J. Smalley
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viii Contents
12. Isolation and Culture of Mouse Pancreatic Islets for Ex Vivo Imaging
Studies with Trappable or Recombinant Fluorescent Probes . . . . . . . . . . . 171
Magalie A. Ravier and Guy A. Rutter
13. Isolation and Culture of Adult Mouse Hepatocytes . . . . . . . . . . . . . . . . 185
Wan-Chun Li, Kate L. Ralphs, and David Tosh
14. Isolation and Culture of Mouse Intestinal Cells . . . . . . . . . . . . . . . . . . 197
Charles Frederick Campbell
15. Derivation of Primary Choroid Plexus Epithelial Cells from the Mouse . . . . . 207
Trevelyan R. Menheniott, Marika Charalambous,
and Andrew Ward
16. The Preparation of Primary Cortical Neuron Cultures and a Practical
Application Using Immunofluorescent Cytochemistry . . . . . . . . . . . . . . 221
Carla Sciarretta and Liliana Minichiello
17. Cell Culture of Primary Cerebellar Granule Cells . . . . . . . . . . . . . . . . . 233
Dana Krämer and Liliana Minichiello
18. Isolation and Generation of Neurosphere Cultures from Embryonic
and Adult Mouse Brain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 241
Henrik Ahlenius and Zaal Kokaia
Subject Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 253
Contributors
HENRIK AHLENIUS • Section of Restorative Neurology, Laboratory of Neural Stem Cell
Biology, Lund Stem Cell Center, Lund, Sweden
LAURA BARBERI • Department of Histology and Medical Embryology, Istituto Pasteur-
Fondazione Cenci Bolognetti, Interuniversity Institute of Myology, Sapienza University of
Rome, Rome 00161, Italy
PAUL J.R. BARTON • Imperial College London, National Heart & Lung Institute, Heart
Science Centre, Harefield UB9 6JH, UK
NIGEL J. BRAND • Imperial College London, National Heart & Lung Institute, Heart
Science Centre, Harefield UB9 6JH, UK
ZOË D. BURKE • Department of Biology and Biochemistry, Centre for Regenerative
Medicine, University of Bath, Bath BA2 7AY, UK
RETO CALDELARI • Department of Molecular Dermatology, Institute of Animal Pathol-
ogy and DermFocus Vetsuisse Faculty, University of Bern; CELLnTEC, Advanced Cell Sys-
tems AG, Bern, Switzerland
CHARLES F. CAMPBELL • Departments of Surgery, Centre for Cancer Research and Cell
Biology, Queen’s University of Belfast, Belfast BT12 6BJ, Northern Ireland, UK
MARIKA CHARALAMBOUS • Department of Physiology, Development, and Neuroscience,
University of Cambridge, Cambridge CB2 3EG, UK
JAMIE A. DAVIES • Centre for Integrative Physiology, University of Edinburgh, Edinburgh
EH8 9XD, UK
PIERRE-MARIE DEL MORAL • Saban Research Institute, Childrens Hospital Los Angeles,
Los Angeles, CA 90027, USA
LESLEY M. FORRESTER • John Hughes Bennett Laboratory, Queen’s Medical Research
Institute, University of Edinburgh, Edinburgh EH164TJ, UK
ALASTAIR S. GARFIELD • Department of Pharmacology, University of Cambridge, Cam-
bridge CB2 1PD, UK
MELANY JACKSON • John Hughes Bennett Laboratory, Queen’s Medical Research Insti-
tute, University of Edinburgh, Edinburgh EH164TJ, UK
ELIZABETH A. JONES • Department of Medical Genetics and Regional Genetic Service,
Central Manchester NHS Foundation Trust, St Mary’s Hospital, Manchester M13 0JH, UK
ZAAL KOKAIA • Section of Restorative Neurology, Laboratory of Neural Stem Cell Biology,
Lund Stem Cell Center, Lund, Sweden
DANA KRÄMER • Mouse Biology Unit, European Molecular Biology Laboratory, Montero-
tondo 00015, Italy
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