Methods in cell biology. Vol. 57, Animal cell culture methods

361 Pages·1998·24.51 MB·English

by  Jennie P Mather

Series Editors
Leslie Wilson
Department of Biological Sciences
University of California, Santa Barbara
Santa Barbara, California
Paul Matsudaira
Whitehead Institute for Biomedical Research and
Department of Biology
Massachusetts Institute of Technology
Cambridge, Massachusetts
Methods in Cell Biology
Prepared under the Auspices of the American Society for Cell Biology
VOLUME 57
Animal Cell Culture Methods
Edited by
Jennie P. Mather
Genentech, Inc.
South San Francisco. California
and
David Barnes
Division of Cell, Developmental, and
Molecular Biology/Genetics
American Type Culture Collection
Manassas, Virginia
ACADEMIC PRESS
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Cover photo (combbound): Human Schwann cell in culture.
Immunofluorescent staining of passage 4 cultures for the
marker GFAP (see Chapter 9 for further details).
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CONTRIBUTORS
Numbers in parentheses indicate the pages on which the authors’ contributions begin.
Shahabuddin Alam (69), Laboratory of Cellular Regulation Technology, Gradu-
ate School of Genetic Resources Technology, Kyushu University, Hakozaki,
Higashi-ku, Fukuoka 812-81, Japan
David Barnes (3), Division of Cell, Developmental, and Molecular Biology/
Genetics, American Type Culture Collection, Manassas, Virginia 20110
Kenneth D. Bauer (265), Genentech, Inc., South San Francisco, California 94080
Christopher J. Bayne (187), Department of Zoology and Evironmental Health
Sciences Center, Oregon State University, Corvallis, Oregon 97331
Christopher J. Donahue (265), Genentech, Inc., South San Francisco, Califor-
nia 94080
Michael G. Gabridge (49), University Technology Corporation, Boulder, Colo-
rado 80466
Anna-Katerina Hadjantonakis (279), Samuel Lunenfeld Research Institute,
Mount Sinai Hospital, Toronto, Ontario, Canada M5G 1x5
Robert J. Hay (31), Cell Culture Department, American Type Culture Collection,
Rockville, Maryland 20852
Angela Helmrich (3), Division of Cell, Developmental, and Molecular Biology/
Genetics, American Type Culture Collection, Manassas, Virginia 201 10
Marie-Claude C. Hofmann (93), Department of Biology, The University of Day-
ton, Dayton, Ohio 45469
Bharati Hukku (203), Cell Culture Laboratory, Children’s Hospital of Michigan,
Department of Pediatrics, Wayne State University School of Medicine, Detroit,
Michigan 48201
Joseph Kaplan (203), Cell Culture Laboratory, Children’s Hospital of Michigan,
Department of Pediatrics, Wayne State University School of Medicine, Detroit,
Michigan 48201
Yoshinori Katakura (69, 11l ),L aboratory of Cellular Regulation Technology,
Graduate School of Genetic Resources Technology, Kyushu University, Hako-
zaki, Higashi-ku, Fukuoka 812-81, Japan
Ronghao Li (167), Signal Pharmaceuticals, Inc., San Diego, California 92121
Carolyn Kay Lincoln (49), Bionique Testing Laboratories, Inc., Saranac Lake,
New York 12983
Deryk T. Loo (251), Bristol-Myers Squibb Pharmaceutical Research Institute,
Princeton, New Jersey 08543
Jennie P. Mather (19,219,265), Genentech, Inc., South San Francisco, Califor-
nia 94080
xi
xii Contributors
Hildegard Meissner (147), Department of Neurosurgery, Laboratory for Brain
Tumor Biology, University Hospital Hamburg Eppendorf, 20246 Hamburg,
Germany
Gary F. Merrill(229), Department of Biochemistry and Biophysics, Oregon State
University, Corvallis, Oregon 97331
Jose Luis Millan (93), The Burnham Institute, La Jolla Cancer Research Center,
La Jolla, California 92037; and Department of Medical Genetics, Umed Univer-
sity, Umei, Sweden
Alison Moore (265), Amgen, Inc., Thousand Oaks, California 91320
Andrhs Nagy (279), Samuel Lunenfeld Research Institute, Mount Sinai Hospital,
Toronto, Ontario, Canada M5G 1x5
David M. Phillips (297), The Population Council, New York, New York 10021
Melinda Pirity (279), Samuel Lunenfeld Research Institute, Mount Sinai Hospi-
tal, Toronto, Ontario, Canada M5G 1x5
Jill R. Rillema (251), Bristol-Myers Squibb Pharmaceutical Research Institute,
Princeton, New Jersey 08543
Sanetaka Shirahata (69, lll), Laboratory of Cellular Regulation Technology,
Graduate School of Genetic Resources Technology, Kyushu University, Hako-
zaki, Higashi-ku, Fukuoka 812-81, Japan
Kiichiro Teruya (1l l), Laboratory of Cellular Regulation Technology, Graduate
School of Genetic Resources Technology, Kyushu University, Hakozaki,
Higashi-ku, Fukuoka 812-81, Japan
Yu-li Wang (313), Cell Biology Group, Worcester Foundation for Biomedical
Research, Shrewsbury, Massachusetts 01545
Manfred Westphal (147), Department of Neurosurgery, Laboratory for Brain
Tumor Biology, University Hospital Hamburg Eppendorf, 20246 Hamburg,
Germany
Sally P. Wheatley (313), Cell Biology Group, Worcester Foundation for Biomedi-
cal Research, Shrewsbury, Massachusetts 01545
Teresa K. Woodruff (333), Northwestern University, Departments of Medicine
and Neurobiology and Physiology, Chicago, Illinois 60611
PREFACE
In this volume we provide a broad introduction to animal cell culture tech-
niques and applications. We also try to integrate into this presentation the concep-
tual framework from which the techniques are derived. The book is divided
into four sections: (I) Principles of Cell Culture; (11) Establishing Cell Lines;
(111) Specialized Culture Techniques; and (IV) Microscopy and Morphology. In
each case, detailed technical treatment is balanced with the succinctness necessary
to contain the work to a single volume. It is our hope that each section and
chapter provide sufficient information to enlighten a reader new to the field
while providing direction to additional sources of information in the primary
literature as well as to relevant previous volumes of this series.
The first section is devoted to the basics of cell culture: laboratory setup,
critical skills, and information with which to make choices of the appropriate
cell culture system. Particular attention is given to the rationale behind routine
cell culture approaches. This includes issues such as why and how commonly
used cell culture medium formulations were developed and the advantages and
disadvantages of using both familiar cell lines and some less frequently used
lines. Our intention is to provide the reader with a basis for troubleshooting as
well as for choosing the best system for the intended purpose. In some cases
the best choice may not be the system that is most commonly used or easiest
to propagate.
An investigator exploring cell culture models should not presume that the
possibilities are limited to available cell lines. In Section I1 we present techniques
and rationale for developing cell culture model systems to fit individual needs.
Specific examples have been chosen to illustrate general principles, and several
of the more common and useful cell culture manipulations are presented. These
include introduction and expression of exogenous DNA (transfection), cell fusion
(hybridoma derivation), cell line characterization (e.g., karyotyping), serum-free
cell culture, and derivation of cell lines of human origin.
Section 111, on specialized culture techniques, provides direction in laboratory-
scale culture for isolation of recombinant products, as well as techniques for
manipulation and measurement of cell proliferation and death, the cell cycle,
and cell differentiation. Emphasis is also placed on cell culture techniques and
applications associated with embryonal stem cells.
The last section points out the critical role of light and electron microscopy
in cell-culture-related work. Also included in this section are various means for
cellular-molecular localization of both proteins and nucleic acids.
xiii
XlV Preface
The editors thank the contributors to this volume for their contributions and
the editors of the series for helpful suggestions. We hope this volume not only
will provide a starting point for new researchers in the field who wish to apply
cell culture techniques to their particular scientific interests, but also will provide
useful additional information and viewpoints to those already expert in cell
culture methodology.
Jennie P. Mather
David Barnes
SECTION I
Principles of Cell Culture
The chapters in the first section are designed to present a brief review
of the basic principles of cell culture. The first chapter provides guidance
for those who are setting up a tissue culture facility or a tissue culture
space in their laboratory. Many people lose a great deal of time and
expend unnecessary effort through not taking sufficient time and thought
to choose the correct cell type, medium, and culture configuration to
achieve their goals. Chapter 2 reviews the role of tissue culture media in
an in vitro system and the different media that are available, whereas
Chapter 3 reviews the cell lines available and the culture repositories
where they can be obtained. Finally, Chapter 4 reviews the different
types of physical, chemical, and biological contamination that can destroy
experiments and/or cause artifactual results. Gross bacterial or fungal
contamination is by far the easiest type of contamination to deal with
because it is so obvious. However, chemically contaminated media or
mycoplasma contamination can be difficult to detect yet cause real prob-
lems in a cell culture laboratory, leading to invalid experimental data.
Section I should provide a good introduction to the special aspects of
2 Section I
laboratory practice that are unique to cell culture and a good review of
the most up to date information in these areas.
The following chapters deal with specific aspects of cell culture that
are deemed to be most important to the cell or molecular biologist wishing
to use cell culture as a tool in hidher work. Many of the chapters emphasize
general principles that will help the investigator select the appropriate
and most efficient tools to reach a desired goal, such as localizing a specific
protein, scaling up cell culture, or establishing a cell line from a normal
or transformed cell. The reader is referred to other volumes in this series,
where applicable, for more detailed protocols concerning some of the
individual techniques discussed here.
CHAPTER 1
Animal Cell Culture Equipment
and Techniques
Angela Helmrich and David Barnes
Division of Cell, Developmental, and Molecular Biology/Genetics
American Type Culture Collection
Manassas, Virginia 201 10
~~
I. Introduction
11. Equipment
A. Hoods
B. Incubators
C. Microscopes
D. Autoclaves
E. Water Purification Equipment and Medlum Filtration Devices
F. Cell Counter
G. Liquid Nitrogen Storage Tanks
H. Water Baths, Centrihges, Freezers, and Refrigerators
111. Laboratory Design
IV. Materials
A. Reagents, Media, and Serum
B. Cell Culture Plasticware and Glassware
V. Cell Culture Methods
A. Sterile Technique and Routine Procedures
B. Primary Culture
C. Multipassage Culture and Cloning
D. Freezing Cells
References
I. Introduction
This chapter is devoted to some of the basics of cell culture equipment and
techniques. It is based largely on innovations, observations, realizations, acci-
METHODS IN CELL BIOLOGY. VOL. 57 3
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