Table Of ContentMammalian Cell Culture
The Use of Serum-Free
Hormone-Supplemented Media
Matntnalian Cell Culture
The Use of Serum-Free
Hormone-Supplemented Media
Edited by
Jennie P. Mather
The Population Council and The Rockifeller University
New York, New York
Plenum Press· NewY ork and London
Library of Congress Cataloging in Publication Data
Main entry under title:
Mammalian cell culture.
Bibliography: p.
Includes index.
1. Cell culture. 2. Culture media (Biology). 3. Mammals-Cytology. I. Mather,
Jennie P., 1948- . II. Title: Semm-free hormone supplemented media. [DNLM:
1. Culture media. 2. Cells, Cultured. QS 530 M265]
QH585.M36 1984 599'.087 84-1985
ISBN-\3: 978-1-4615-9363-8 e-ISBN-I3: 978-1-4615-9361-4
001: 10.10071978-1-4615-9361-4
© 1984 Plenum Press, New York
Softcover reprint of the hardcover 1 st edition 1984
A Division of Plenum Publishing Corporation
233 Spring Street, New York, N.Y. 10013
All rights reserved
No part of this book may be reproduced, stored in a retrieval system, or transmitted,
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Contributors
David Barnes - Department of Biological Sciences, University of
Pittsburgh, Pittsburgh, Pennsylvania 15260 '
Theodore R. Breitman - Laboratory of Tumor Cell Biology, N a
tional Cancer Institute, National Institutes of Health, Bethesda,
Maryland 20205
Brian I. Carr - Division of Cytogenetics and Cytology and Depart
ment of Medical Oncology, City of Hope National Medical Center,
Duarte, California 91010
Paul V. Cherington - Laboratory of Neoplastic Disease Mechanisms,
Dana-Farber Cancer Institute, Boston, Massachusetts 02115
J. E. Estes - Department of Pharmacology, Cancer Cell Biology
Program, Cancer Research Center, University of North Carolina
School of Medicine, Chapel Hill, North Carolina 27514
Izumi Hayashi - Division of Cytogenetics and Cytology and De
partment of Medical Oncology, City of Hope National Medical
Center, Duarte, California 91010
Hiromichi Hemmi - Laboratory of Tumor Cell Biology, National
Cancer Institute, National Institutes of Health, Bethesda, Maryland
20205
P. H. Howe - Department of Pharmacology, Cancer Cell Biology
Program, Cancer Research Center, University of North Carolina
School of Medicine, Chapel Hill, North Carolina 27514
Douglas M. Jefferson - Department of Molecular Pharmacology,
Albert Einstein College of Medicine, Bronx, New York 10461
Beverly R. Keene - Laboratory of Tumor Cell Biology, National
Cancer Institute, National Institutes of Health, Bethesda, Maryland
20205
v
vi Contributors
E. B. Leof • Department of Pharmacology, Cancer Cell Biology
Program, Cancer Research Center, University of North Carolina
School of Medicine, Chapel Hill, North Carolina 27514
Jennie P. Mather • The Population Council and The Rockefeller
University, New York, New York 10021
W. J. Pledger • Department of Pharmacology, Cancer Cell Biology
Program, Cancer Research Center, University of North Carolina
School of Medicine, Chapel Hill, North Carolina 27514
Lola M. Reid • Department of Molecular Pharmacology, Albert Ein
stein College of Medicine, Bronx, New York 10461
Ginette Serrero-Dave • Cancer Center, University of California, San
Diego, La Jolla, California 92093. Present address: W. Alton Jones
Cell Science Center, Lake Placid, New York 12946
Mary Taub • Biochemistry Department, State University of New
York at Buffalo, Buffalo, New York 14214
Stephen D. Wolpe • The Population Council, New York, New York
10021
Preface
The advantages of obtaining a completely defined environment for the
growth of cells in vitro were recognized very early in the history of cell
culture (Lewis and Lewis, 1911). Continued interest in the nutritional
requirements of cells in vitro and in providing an optimal environment
for cells led to the development of the complex nutrient mixtures
available today in many media (Waymouth, 1972; Ham, 1965).
However, serum remained an essential component of medium for
the growth of most cell types in culture. The question of what factor
(or factors) in serum was essential for cell growth and survival remained
unanswered for several decades. Initially, experiments were designed
to purify the "active component" of serum for the growth of cells in
culture. These experiments identified fetuin (Fisher et at., 1958) and
nonsuppressible insulinlike activity (Temin et at., 1972) as important
components of serum. However, the complexity of serum and the very
low levels of active components in serum hindered progress in identi
fying and isolating serum factors.
In retrospect, the analytical approach can be seen to be extremely
difficult due to the complex interactions of various hormones and
growth factors and the requirements of cells for several very different
types of hormones for growth and function. However, these experi
ments and the development of hormone-dependent cell lines (Clark et
at., 1972; Armelin, 1973; Nishikawa et at., 1975) led to the hypothesis
that the role of serum in cell culture was to provide a complex of
hormones that were growth-stimulatory for given cell types (Sato, 1975).
The first experimental evidence in support of this hypothesis was
provided when the GH3 pituitary cell line was grown in serum-free
medium supplemented with hormones, growth factors, and transferrin
(Hyashi and Sato, 1976).
vii
viii Preface
Subsequently, the development of hormone-supplemented serum
free conditions for the growth of several cell lines originating from
different tissues (Bottenstein et at, 1979; Mather and Sato, 1979; Barnes
and Sato, 1981) allowed several generalizations concerning the growth
of cells in serum-free medium. (1) Serum can be replaced by a mixture
of hormones, growth factors, transport proteins, vitamins, and attach
ment factors. (2) The requirements for these factors differ for different
cell types. (3) The requirements for cell lines derived from the same
cell type are similar or identical for cells from different species. (4) The
factors used to grow a cell line derived from a particular cell type can
be used for primary cultures of that cell type. These primary cultures
will frequently exhibit improved growth andlor function compared with
those in serum-supplemented culture. (5) Serum-free culture can be
used to avoid fibroblast overgrowth in primary cultures and select for
specific cell types. (6) Functional cell lines can be established in serum
free medium from primary cultures of cell types not previously estab
lished in culture.
Since the first report of the growth of GH3 cells in 1976, this
approach has been widely used by investigators in a number of areas
(Sato et at., 1982). It should be emphasized, however, that the use of
hormone-supplemented serum-free culture is more than a new tech
nique. It involves some very basic changes in how we view in vitro
cultures of eukaryotic cells. These cells can no longer be viewed simply
as a microorganism (Puck, 1972), but rather as part of a complex
interactive system involving matrix substrate, cell-cell contact, and cell
cell communication via soluble factors. It is also apparent that many
aspects of cell behavior in vitro that led some investigators to consider
this an artifactual system are, in fact, responses to an inappropriate
environment (i.e., serum and plastic surfaces). With the use of appro
priate hormone-supplemented media and substrates, it can be seen that
cell lines, even those that have been in culture for many years, can
express cell-specific functions and respond to hormones in a physiologic
manner. This is, perhaps, the greatest power of defined cell culture.
The synthetic approach, by defining step-by-step the conditions re
quired for cell growth or function, gives deep insight into the com
plexity of the regulation of cell physiology in vivo, which is impossible
to obtain by other means.
This book makes no attempt to cover the entire field of hormone
supplemented cell culture. Rather, the contributions have been chosen
to illustrate several of the diverse areas of research that have benefited
from this new view of cell physiology. By giving in-depth coverage to
these areas and the potential of using serum-free culture to define new
Preface ix
areas of research, it is hoped that the reader will gam a deeper
appreciation of the possibilities of this approach.
Studies on the role of hormones in controlling the initiation of cell
proliferation and cell cycle (Chapters 1 and 2) give more insights into
the physiologic regulation of cell growth than was possible using other
methods of cell synchronization such as amino acid starvation. The use
of serum-free culture has also allowed studies of differentiation of cells
such as adipocytes (Chapter 3) and myelomonocytes (Chapter 4) in
defined conditions. Cell-produced products can be more easily purified
from serum-free culture. This is especially useful in monoclonal anti
body production (Chapter 5). The regulation of physiologic function
of cells can be studied (Chapter 6). The complex interactions of cells
in vivo in response to extraneous agents such as chemical carcinogens
(Chapter 7), hormones (Chapter 6), or factors produced locally in an
organ (Chapter 8) can be studied using in vitro defined culture systems.
The role of defined attachment factors (Chapter 9) or isolated biomatrix
(Chapter 10) can be studied in vitro in defined systems to increase our
understanding of the role of the basal lamina and cell-substrate
interactions in vivo.
The complex nature of hormone action and interactions in vitro is
immediately apparent from these studies. Thus, a hormone may act
directly on a cell in vitTO to stimulate progression through the cell cycle
or specific cell function. A hormone might also act indirectly by
stimulating the secretion of a cell-produced attachment factor or inhibit
the production of enzymes such as collagenase, which might degrade a
cell-produced attachment factor. The increased levels of such factors
might then lead to increased cell growth or function. A hormone might
also act by increasing the secretion of a transport protein such as
transferrin, which would then transport a required nutrient into the
cell and increase cell growth. A hormone might increase the production
of a growth factor, which would then act to stimulate growth in that
cell or inhibit the production of a growth inhibitor. Given these and
other possible modes of action and interaction discussed in these
chapters, it is apparent that the regulation of cell growth and function
by a few hormones, growth factors, transport factors, and attachment
factors can be a very complex matter indeed. However, these problems
can be approached using defined culture. The next few decades should
prove to be a period of exciting advances in our understanding of cell
physiology in vitTO and in vivo.
It is appropriate to express the appreciation of the author for
many conversations with Dr. Gordon Sato. Without his vision, gener-
x Preface
osity, and support, much of the work in this field, and this book, would
not have been possible. I would also like to thank Ms. Catherine
Galloway for her editorial assistance.
Jennie P. Mather
References
Armelin, H. A., 1973. Pituitary extracts and steroid hormones in the control of 3T3 cell
growth, Proc. Natl. A cad. Sci. U.S.A. 70:2702-2706.
Barnes, D., and Sato, G., 1981, Serum-free cell culture: A unifying approach, Cell 22:69-
655.
Bottenstein, J. E., Sato, G. H., and Mather, J. P., 1979, Growth of neuroepithelial derived
cell lines in serum-free hormone-supplemented media, in: Hormones and Tissue Culture
(G. Sato and R. Ross, eds.), Cold Spring Harbor Press, Cold Spring Harbor, New
York, pp. 531-544.
Clark, J. L., jones, K. L., Gospodarowicz, D., and Sato, G. H., 1972, Growth response to
hormones by a new rat ovary cell line, Nature New Bio. 236: 180-181.
Fisher, H. W., Puck, T. T., and Sato, G., 1958, Molecular growth requirements of single
mammalian cells: The action of fetuin in promoting cell attachment to glass, Proc.
Natl. Acad. Sci. U.S.A. 44:4-10.
Ham, R. G., 1965, Clonal growth of mammalian cells in a chemically defined synthetic
medium, Proc. Natl. Acad. Sci. U.S.A. 53:288-293.
Hayashi, I., and Sato, G., 1976, Replacement of serum by hormones permits the growth
of cell in a defined medium, Nature (London) 159:132-134.
Lewis, M. R., and Lewis, W. H., 1911, The cultivation of tissues from chick embryos in
solutions of NaC!, CaCI2, KCl, and NaHCOs, Anat. Rec. 5:277-293.
Mather, J. P., and Sato, G., 1979, The growth of mouse melanoma cells in serum-free
hormone supplemented medium, Exp. Cell Res. 120:191-200.
Nishikawa, K., Armelin, H. A., and Sato, G., 1975, Control of ovarian cell growth in
culture by serum and pituitary factors, Proc. Nat!. Acad. Sci. U.S.A. 72:483-487.
Puck, T. T., 1972, The Mammalian Cells as a Microorganism, Holden-Day, Inc., San Francisco.
Sato, G., 1975, The role of serum in cell culture, in: Biochemical Action of Homwnes, Volume
III (G. Litwak, ed.), Academic Press, Inc., New York, pp. 391-396.
Sato, G. H., Pardee, A. B., and Sirbasku, D. A., 1982, G1'Owth of Cell in Hormonally Defined
Media, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York.
Temin, H., Pierson, R. W.,jf., and Dulak, N. c., 1972, The role of serum in the control
of multiplication of avian and mammalian cells in culture, in: Growth, Nutrition and
Metabolism of Cells in Culture, Volume I (G. H. Rothblat and V. J. Cristafalo, eds.),
Academic Press, New York, pp. 49-81.
Waymouth, c., 1972, Construction of Tissue Culture Media, in: GTOwth,Nutrition and
Metabolism of Cells in Culture, Volume I (G. H. Rothblatt and V. J. Cristofalo, eds.),
Academic Press, New York, pp. 11-49.
Contents
CHAPTER 1
Serum Factor Requirements for the Initiation of Cellular
Proliferation
W. J. PLEDGER, J. E. ESTES, R. H. HOWE,
and E. B. LEOF
Introduction .................................... 1
Multiple Serum Components Required for Cellular
Proliferation .................................... 2
EGF and Somatomedin C Can Replace the GO/G Progession
1
Activity of PPP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
EGF and Somatomedin C Reduce the Minimum G Transit
1
Time .......................................... 5
A vail ability of Free Somatomedin C Controls Minimum G
1
Transit Time. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Somatomedin C Regulates Progression of Late G Phase and
1
Commitment to DNA Synthesis ...................... 7
EGF Is Required during Traverse of Early G 9
1 • . • • • • . • • • .
Summary and Conclusions . . . . . . . . . . . . . . . . . . . . . . . . .. 11
References ...................................... 13
xi
