Table Of ContentContributors
DONALD ARMSTRONG
MICHAEL F. BARILE
LEWIS L. CORIELL
ETIENNE de HARVEN
JÖRGEN FOGH
NELDA B. HOLMGREN
GEORGE E. KENNY
ALEXIS J. KNIAZEFF
DONALD J. MERCHANT
F. KINGSLEY SANDERS
CYRIL S. STULBERG
Contamination in
Tissue Culture
Edited by
JORGEN FOGH
Sloan-Kettering Institute for Cancer Research
Rye, New York
ACADEMIC PRESS New York and London 1973
A Subsidiary of Harcourt Brace Jovanovich, Publishers
COPYRIGHT © 1973, BY ACADEMIC PRESS, INC.
ALL RIGHTS RESERVED.
NO PART OF THIS PUBLICATION MAY BE REPRODUCED OR
TRANSMITTED IN ANY FORM OR BY ANY MEANS, ELECTRONIC
OR MECHANICAL, INCLUDING PHOTOCOPY, RECORDING, OR ANY
INFORMATION STORAGE AND RETRIEVAL SYSTEM, WITHOUT
PERMISSION IN WRITING FROM THE PUBLISHER.
ACADEMIC PRESS, INC.
Ill Fifth Avenue, New York, New York 10003
United Kingdom Edition published by
ACADEMIC PRESS, INC. (LONDON) LTD.
24/28 Oval Road, London NW1
Library of Congress Cataloging in Publication Data
Fogh,Jorgen.
Contamination in tissue culture.
Includes bibliographies.
1. Culture contamination (Biology) 2. Tissue
culture. I. Title. [DNLM: 1. Histological technics.
2. Tissue culture. QS530 F655c 1973]
QH585.F64 574.8 '2 '0724 72-88363
ISBN 0-12-261850-5
PRINTED IN THE UNITED STATES OF AMERICA
List of Contributors
Numbers in parentheses indicate the pages on which the authors' contributions begin.
DONALD ARMSTRONG (51), Department of Medicine, Infectious Desease
Service and Microbiology Laboratory, Memorial Hospital for Cancer
and Allied Diseases, Cornell University Medical College, New York,
New York
MICHAEL F. BARILE (131), Section of Mycoplasma, Laboratory of Bacterial
Products, Division of Biologies, National Institutes of Health, Depart-
ment of Health, Education, and Welfare, Bethesda, Maryland
LEWIS L. CORIELL (29), Institute for Medical Research, Camden, New
Jersey
ETIENNE de HARVEN (205), Sloan-Kettering Institute for Cancer Research,
New York, New York
JORGEN FOGH (65, 173), Sloan-Kettering Institute for Cancer Research,
Rye, New York
NELDA B. HOLMGREN (195),* The W. Alton Jones Cell Science Center of the
Tissue Culture Association, Inc., Lake Placid, New York
GEORGE E. KENNY (107), Department of Pathobiology, School of Public
Health and Community Medicine, University of Washington, Seattle,
Washington
ALEXIS J. KNIAZEFF (233), Department of Medicine, School of Medicine,
University of California, San Diego, California
* Present address: Park Place Townhouses, 1170 Mt. Mary Drive, Green Bay, Wisconsin
IX
χ List of Contributors
DONALD J. MERCHANT (257),* W. Alton Jones Cell Science Center of the
Tissue Culture Association, Inc., Lake Placid, New York
F. KINGSLEY SANDERS (243), Division of Cell Biology, Sloan-Kettering
Institute for Cancer Research, Rye, New York
CYRIL S. STULBERG (1), The Child Research Center of Michigan, The Child-
ren's Hospital of Michigan, Detroit, Michigan
* Present address: Department of Microbiology and Cell Biology, Eastern Virginia Medical
School, Norfolk, Virginia
Preface
Bacteria, fungi, mycoplasmas, viruses, parasites, and also cells of foreign
types or species may accidentally be introduced into tissue cultures. The high
susceptibility of tissue cultures to bacteria and fungi and their detri-
mental effects on the cells were realized from the earliest days of this
discipline. However, the problems related to other types of contaminants
only became obvious during the last two decades in which tissue cultures
evolved as a commonly used "tool" in biological research. Still, some apathy
toward these problems has prevailed, and much work has been carried out
with contaminated cell systems leading to incorrect interpretations of
diagnostic and experimental data. The purpose of this book is to summarize
and discuss presently available information on tissue culture contamination
and, hopefully, stimulate a greater awareness of and interest in its many
aspects. This is the first comprehensive volume on this subject.
The practical aspects concern the sources, the prevention, the detection,
and the elimination of contaminants. Data on their frequency of occurrence
and relative importance are presented. In addition, the many different effects
on cultured cells, sometimes severe and not infrequently irreversible, are
described and discussed as they are viewed by the cytologist, bacteriologist,
geneticist, and virologist. Some of these effects were only recognized
recently. Emphasis has been given to the academic aspects of the subject, and
although techniques are included, this book is not intended to be a manual
on contamination. Some problems have not as yet been settled. Views by
different contributors may not necessarily agree. Yet, it is hoped that this
volume will meet the needs of investigators, teachers, students, and techni-
cians within the many disciplines of cell biology, physiology, cytology,
virology, immunology, genetics, oncology, molecular biology, biochemistry,
and biophysics in which tissue and cell cultures are used, either as the
primary object of research or as tools.
xi
xii Preface
There is an analogy between tissue culture contamination and the pollu-
tion of the environment of man, animals, and plants. Actually, our labora-
tory problems are just part of the overall perplexity. Pressures for increased
production and strong competition have tended to reduce the quality of pro-
cedure characteristic of the small work load and the individual approach.
However, this volume shows that there are means toward solving most of the
tissue culture problems. If future trends in research will be toward greater
attention to quality control of the cell systems employed, the effort that
went into this book will not have been needless.
The authors were selected for their particular competence and, in most
cases, extensive experience in special aspects of the subject. A substantial
amount of work went into the preparation of the chapters, and for this I am
grateful to all the contributors. I am particularly indebted to the staff of
Academic Press for their effort. A special thanks to Shirley DeVore, my
secretary, for her great help in attending to the many necessary details, and to
Judith Berens with whom she prepared the Subject Index.
JORGEN FOGH
CHAPTER
Extrinsic Cell Contamination
of Tissue Cultures
CYRIL S. STULBERG
I. Introduction 2
A. Modern Development of Cell Culture 2
B. The Problem of Extrinsic Cell Contamination 2
C. Problems of Cell Identification 3
II. Interspecies Identification of Cell Cultures 4
A. General Considerations 4
B. Cytogenetic Methods 5
C. Cytotoxicity 7
D. Hemagglutination 7
E. Mixed Agglutination 8
F. Fluorescent Antibody 9
G. Other Immunologic Methods 14
H. Stable and Variable Antigenic Markers 14
I. Isoenzymes 15
J. Malignancy 15
III. Intraspecies Identification of Cell Cultures 16
A. Cytogenetic Analysis 16
B. Metabolic Markers of Genetic Origin ". 16
C. Isoenzyme Profiles 17
D. Immunology 20
E. Fusion and Hybridization 21
IV. Current Status of Cell Contamination 22
References 23
1
2 Cyril S. Stulberg
I. Introduction
A. MODERN DEVELOPMENT OF CELL CULTURE
For over half a century, investigators have been able to cultivate animal
cells in vitro and their methods have been powerful tools in biology. Develop-
mental highlights of this evolutionary process are the descriptions of long-
term cultures of the L cell line from mice by Earle and his co-workers (Earle,
1943; Evans et al., 1951; Sanford et al., 1948) and the derivation and con-
tinuous propagation of HeLa cell cultures from human malignant tissue by
Gey et al. (1952). Their discoveries allowed animal cells to be handled and
propagated in vitro in a manner similar to microorganisms. A review of the
history of cell culture is beyond the objectives of this chapter. However,
investigators are well aware of the tremendous impact of animal cell cultures
in numerous areas of biology and medicine. As a result of the great expansion
in the use of cell cultures in many areas of research, the question of cell
identification has become increasingly important. In this chapter, the sub-
ject of contamination by extraneous animal cells is emphasized, especially
in view of recent information that has appeared.
B. THE PROBLEM OF EXTRINSIC CELL CONTAMINATION
The advent of both the L and HeLa cell lines and their early applications
to virology (Scherer, 1953; Scherer et al., 1953) together with nutritional
advances, such as that of Eagle (1955), had the effect of bringing forth many
new animal cell lines with additional uses. However, within 10 years it was
evident that along with these developments came other kinds of problems.
Often, cell populations in use during the 1950's were not clearly defined with
regard to their origin or lineage. This occurred partly because decisive or
even adequate methods of identifying cells in vitro were not available. At the
same time that there was a rapid expansion in the use of cell cultures, the pro-
fusion of new lines began to lead to a state of confusion. This was caused in
part by a singular kind of contamination, the inadvertent contamination of
tissue cultures by other animal cells.
Toward the end of the 1950's, accounts of interspecies cell contamination
as determined by cytogenetic or immunologic means began to appear
(Defendi et al., 1958; Rothfels et al, 1959; Brand and Syverton, 1959;
Coombs et al, 1961a; Stulberg et al., 1961a). This was one reason that the
Animal Cell Culture Collection Committee was formed; its purpose was to
provide contaminant-free lines as well as to make available characterized re-
ference cell lines that could be utilized in several areas (Stulberg et al, 1970;
American Type Culture Collection, 1964).
1. Extrinsic Cell Contamination of Tissue Cultures 3
Interspecific cell contamination could be controlled by a variety of pro-
cedures but these methods did not solve the problem of intraspecific con-
tamination. It seemed unreasonable to find that interspecies cell contamina-
tion was widespread and yet not to expect intraspecies contamination to
exist. This worried many investigators and Harris (1964) emphasized that the
latter problem would ultimately have to be understood on a genetic basis and
not on a "modulative" one (see Harris, 1968). Gartler( 1967) initially demon-
strated that when other genetic markers failed to serve the purpose, iso-
enzymes sometimes did, and he also stated that their capacities for identifica-
tion of a cell line would increase depending on the number and kinds of
isoenzyme systems that were available. In fact, recently on the basis of
glucose 6-phosphate dehydrogenase (G6-PD) isoenzymes in cell lines, there
has been a mass of evidence accumulated indicating that most of the human,
permanent, heteroploid lines thought to have independent origins are indeed
derived from a single donor(Gartier, 1967,1968; Peterson, 1968; Petersons
a/., 1968, 1971; Stulberg et αϊ, 1970). Because of such findings, several
problems will have to be reconsidered, among them the phenomenon of
spontaneous malignant transformation in human cell cultures.
C. PROBLEMS OF CELL IDENTIFICATION
Extrinsic contamination of cultures is intimately related to the basic
problem of identification of cells in culture. If a cell culture of a particular
species were to exhibit a function not found in other cultures, an identifica-
tion marker might be established. The hormone-producing cells described by
Sato and his colleagues serve as examples (Yasamuraei a/., 1966). However,
it is important to distinguish between irreversible genetic changes and revers-
ible variations caused by environmental conditions. Harris (1968) cites
evidence showing that once differentiation takes place, the genotype is ir-
reversible but that a modulation is a reversible phenotypic shift occurring in
an unchanging genetic background (also see Puck and Kao, 1968; Franks,
1968; Foley et al., 1965; Fogel, 1968).
A number of irreversible cytogenetic and biochemical characteristics that
have an hereditary basis are tangible means of cell identification in vitro and
range from euploid karyotypes (see Tjio and Levan, 1956; Hsu andBenir-
schke, 1967-1971) to heritable biochemical markers, which have been de-
scribed and summarized by Krooth and Sell (1970). Irreversible changes that
can be induced in cultures are exemplified by the mutant strains produced by
Szybalski and his co-workers (see Szybalski and Szybalska, 1962). However,
well-known reversible (nongenotypic) changes occur readily when many
viruses, and different environmental conditions, are employed.
In the absence of precise identification, cell lines have been designated
