Atlas of Human Tumor Cell Lines

490 Pages·1994·21.523 MB·English

by  Robert K.M. Hay, Jae-GahB. Park and Adi Gazdar (Eds.)

Atlas of
Human Tumor Cell Lines
Edited by
Robert J. Hay
Cell Culture Department
American Type Culture Collection
Rockville, Maryland
Jae-Gahb Park
Department of Surgery
Seoul National University Hospital
Seoul, Korea
Adi Gazdar
Simmons Cancer Center
Southwestern Medical School
Dallas, Texas
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Front cover photograph: Scanning electron micrograph of KATO-III gastric carcinoma
cells. The cultured cells are covered with numerous microvilli, and the typical signet
ring-shaped cells have cave-like depressions on the cell wall. Courtesy of T. Suzuki.
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Library of Congress Cataloging-in-Publication Data
Atlas of human tumor cell lines / edited by Robert J. Hay, Jae-Gahb
Park, Adi Gazdar.
p. cm.
Includes bibliographical references and index.
ISBN 0-12-333530-2
1. Cancer cells—Atlases. 2. Human cell culture—Atlases.
I. Hay, Robert. II. Park, Jae-Gahb. III. Gazdar, Adi F.
[DNLM: 1. Tumor Cells, Cultured-atlases. 2. Tumor Stem Cells-
-atlases. 3. Cell Line—atlases. 4. Neoplasms—pathology-
atlases.]
RC267.A84 1994
616.99'2'0072-dc20 93-25709
CIP
PRINTED IN THE UNITED STATES OF AMERICA
94 95 96 97 98 99 EB 9 8 7 6 5 4 3 2 1
Preface
The utility of human tumor cell lines in culture for a diverse range of research
programs involving modern cellular and molecular biology, genetics, and
oncology is recognized today much more extensively than ever before. Numer-
ous cell lines of interest have been developed and are generally available.
Data on those lines that are both useful and popular accumulate at a breathtak-
ing pace, and the task of remaining up-to-date has become increasingly
difficult. This atlas brings together in a single volume much of the most relevant
information relating to human lines from major tissue categories, including all
of the high-cancer-risk organs. It should be of use not only to those in a broad
range of disciplines working with many different human tumor cell lines in
culture, but also to those searching for continuous human lines with specific
characteristics.
We recognized at the outset that the morphological definition of a cell line is
only one of its characteristics and that this alone is a notoriously flawed tool for
precise identification. In fact, many of the contributing authors point this out
and provide and refer to additional features of specific types of cell lines that
can be used for more positive identification and for further experimentation.
Particulars on cell banking and characterization are included, along with
representative data on the absolute identification of human cell lines by DNA
fingerprinting. Most chapters detail methods for establishment and mainte-
nance, morphology, identification of surface and intracellular antigens, and
secreted products. If pertinent, lists of the available lines and future prospects
for their use in research are included. Phase-contrast and electron photomicro-
graphs are generally supplemented with additional related data on, for exam-
ple, cell line derivation, growth properties in culture, antigenic traits, karyol-
ogy, and related critical genetic attributes such as chromosomal abnormalities
and locations of oncogenes, if known—al I with appropriate reference to recent
publications.
With the exception of several textbooks on cell culture methods, which
generally do not dwell on morphology and other detailed descriptive charac-
teristics of cell lines, only one major volume describing derivation and proper-
ties of many human tumor cell lines has been published [Fogh, J. (ed.) (1975).
xi
xii Preface
"Human Tumor Cells in Vitro." Plenum, New York]. Since publication of that
volume almost two decades ago, there has been a tremendous increase in both
the number and the availability of new lines with unique characteristics. The
Atlas of Human Tumor Cell Lines will fill this gap in information and provide
timely and useful summaries for scientists working in the fields of cell biology
and cancer research.
Robert J. Hay
Jae-Gahb Park
Adi Gazdar
1
Quality Control
and Characterization
of Cell Lines
Robert J. Hay
Cell Culture Department, American Type Culture Collection
Rockville, Maryland 20852
I. Introduction 1 IV. Cellular Cross-
Contamination 8
II. Seed Stock Concept 2 A. Species Verification ί
B. Intraspecies Cross-
Contamination 9
III. Microbial Contamination 3
V. Origin and Function 13
A. Bacteria and Fungi 4
VI. Conclusions 14
B. Mycoplasma Infection 4
C. Viruses 5 References 14
I. Introduction
The scientific literature documents over 250 instances of cross-
contamination in cell culture systems (Nelson-Rees, 1978; Nelson-Rees et
al., 1981; Hukku et al., 1984), and many more have certainly gone unre-
ported. The novice technician or student using cell culture techniques soon
is made painfully aware of the potential for bacterial and fungal infection.
Generally, however, one must be alerted to the more insidious problems of
animal cell cross-contaminations, the presence of mycoplasma, and espe-
cially the potential for latent or otherwise inconspicuous viral infection. The
financial losses in research and production efforts resulting from the use of
contaminated cell lines is incalculable but certainly equivalent to many mil-
lions of dollars. Accordingly, frequent reiteration of the details of cell culture
contaminations and of precautionary steps to avoid and detect such prob-
lems clearly is warranted.
This chapter includes comments on quality control methods applied to
authenticate cell lines, that is, to insure absence of microbial, viral, and cel-
lular contamination, as well as potential tests to verify the identity of human
cell lines for which previous data are readily available. The approach sug-
gested has been developed during establishment of a national cell reposi-
Atlas of Human Tumor Cell Lines
Copyright © 1994 by Academic Press, Inc. All rights of reproduction in any form reserved. 1
2 Robert J. Hay
tory. Specific rationales for applying the tests indicated are included in this
chapter or are discussed in more detail elsewhere (Hay, 1992). All these
tests provide information supplementary to the primarily morphological data
customarily presented in an atlas, such as this one, on human tumor cell
lines.
Most established cell lines have been characterized by the originator and
collaborators well beyond the steps essential for quality control. Specific
details are provided in each subsequent chapter of this book and include, for
example, phase contrast and ultrastructural morphologies; detailed cy-
togenetic analysis; definition of proto-oncogene, oncogene, or oncogene
product presence, nature, and location; detailed evaluation of intermediate
filament proteins; and demonstration of tissue-specific antigens or produc-
tion of other specific products. These characterizations obviously increase
the value of each cell line for research and, perhaps for production work.
However, cell banking organizations need not attempt to repeat all these
tests before distributing the stock cultures. Decisions must be made to estab-
lish the most acceptable authentication steps, consistent with maintaining
the lowest possible cost, to provide a high quality cell stock. Authentication
can be considered the act of confirming or verifying the identity of a specific
line, whereas characterization is the definition of the many traits of the cell
line, some of which may be unique and also may serve later to identify or
authenticate that line specifically. Essential steps for quality control will vary
with the type of cell bank constructed; such minimal descriptive data fre-
quently will be supplemented with a much broader characterization base for
each particular cell line.
II. Seed Stock Concept
Definitions of public repository seed stocks may vary from those used for
specific applications such as production of vaccines or other biologicals. A
scheme illustrating the steps involved in developing the seed stocks is pre-
sented in Fig. 1.
Generally, starter cultures or ampules are obtained from the originator and
progeny are propagated according to instructions to yield the first "token"
freeze. Cultures derived from such token material then are tested for bacte-
rial, fungal, and mycoplasmal contamination. The species of each cell line is
verified. These quality control steps are the minimum ones that must be per-
formed before eventual release of a line. If these tests confirm that further
efforts are warranted, the material is expanded to produce the seed and
distribution stocks. Note that, under ideal conditions, additional major quality
control and characterization efforts are applied to cell populations from seed
stock ampules. Test results refer to specific numbered stocks. The distribu-
1 Quality Control and Characterization 3
Starter Culture
Contamination Checks ±■
Species Verification
Token Freeze
Percent Recovery
Contamination Checks 1 *
Culture to
Species Verification
Isoenzymology Originator for
Karyology Seed | Verification
Clone Forming Efficiency Stock ■
DNA Fingerprinting
Credentials to
Tumorigen icity Tests
Advisors
Retest for Contamination Distribution
Verify Species Stock
Fig. I. A quality control scheme suggested for the addition (acces-
sioning) of a cell line to a collection. Representative tests for token,
seed, and distribution stocks are indicated (left), as are steps for
review by originating investigators and advisors (right). Additional
characterizations such as general or specific screens for viruses,
functional tests, and definitions of particular inclusions or ultrastruc-
ture may be applied when critical, or with special support. Immuno-
logical tests are required, especially to identify specific protein in-
clusions or other products such as immunoglobulins released by
hybridomas.
tion stock consists of ampules that are distributed on request to investigators.
The reference seed stock, on the other hand, is retained to generate further
distribution stocks as the initial distribution stock becomes depleted. The
degree of characterization applied to master cell banks or master working
cell banks in production facilities is generally more rigorous, although the
seed stock, like the master cell bank, is used as a reservoir to replenish
depleted distribution lots over the years. By adherence to this principle,
problems associated with genetic instability, cell line selection, senescence,
or transformation can be avoided.
III. Microbial Contamination
Microbial contamination in cell culture systems remains a serious problem.
Cryptic contaminants, even of readily isolatable bacteria and fungi, are
missed by many laboratories. The American Type Culture Collection (ATCC)
still receives cultures, even for the patent depository, that contain yeast, fil-
amentous fungi, and/or mycoplasma contaminants.
4 Robert J. Hay
A Bacteria and Fungi
Microscopic examination is only sufficient for detection of gross contami-
nations; even some of these cannot be detected readily by simple observa-
tions. Therefore, an extensive series of culture tests also is required to pro-
vide reasonable assurance that a cell line stock or medium is free of fungi
and bacteria (Hay, 1992).
B. Mycoplasma Infection
Contamination of cell cultures by mycoplasma can be a much more insidi-
ous problem than that created by growth of bacteria or fungi. Although the
presence of some mycoplasma species may be apparent because of the
degenerative effects induced, other mycoplasmas metabolize and proliferate
actively in the culture without producing any overt morphological change in
the contaminated cell line. Thus, cell culture studies relating to metabolism,
surface receptors, virus-host interactions, and so forth are certainly suspect
in interpretation, if not negated entirely, when conducted with cell lines that
harbor mycoplasma. The seriousness of these problems can be documented
through published data from testing services and cell culture repositories.
The high incidence of mycoplasma contamination from human operators is
supported by the fact that Mycoplasma orale and others of human origin
(Mycoplasma hominus, Mycoplasma salivarium, and Mycoplasma fermen-
tans) are among those most frequently isolated. In the study by Del Giudice
and Gardella (1984), of the 34,697 lines tested, 3955 (11%) were positive;
36% of these isolates were mycoplasmas of human origin (Table I). A high
incidence of isolation of Mycoplasma hyorhinus was noted, that may result
from use of contaminated sera or by culture-to-culture spread in laboratories
working with infected biologicals. After a more recent study, Uphoff et al.
(1992) reported that 84 (33%) of 253 cell lines submitted for their developing
cell repository in Germany were infected with mycoplasma. A comparative
analysis of the sensitivity of six different detection methods was conducted,
Table I
Mycoplasma Isolation, 1966-1982a
Number of specimens 34,697
Number of positives^ 3,955
Mycoplasmas of human origin (% of total) 36
Mycoplasmas of bovine origin (% of total) 31
a Summarized from Del Giudice and Gardella (1984).
About 14% of these were infected with more than one
species of mycoplasma.
1 Quality Control and Characterization 5
with the recommendation that a combination of two or three be selected for
any routine testing regimen.
Nine general methods are available to detect mycoplasma. The direct cul-
ture test and the indirect test employing a bis-benzimidazole fluorochrome
stain (Hoechst 33258) for DNA are used routinely in many laboratories to
check incoming cell lines and all working cell stocks. Hay ei al. (1989) re-
viewed detection and elimination methods. Advanced techniques involving
the polymerase chain reaction (PCR) provide the most sensitive methods for
detection (Spaepen etal., 1992; Uemori etal., 1992).
Four general recommendations can be offered to avoid mycoplasma infec-
tion. The implementation of an effective regime to monitor cell lines for myco-
plasma is one critical step. Quarantining all new untested lines and using
mechanical pipetting aids are others. Most experts also strongly suggest that
the use of antibiotics be eliminated when possible. Antibiotic-free systems
permit overgrowth by bacteria and fungi to provide ready indication when-
ever a lapse in aseptic technique occurs. When the initial tissue is used, for
example, a human tumor sample, antibiotics may be employed intially, but
after the primary population has grown out and been cryopreserved, reconsti-
tuted cells should be propagated further in antibiotic-free medium.
C. Viruses
Of the various tests to detect adventitious agents associated with cultured
cells, those detecting endogenous and contaminant viruses are the most
problematic. Representative viruses possibly present in human and other
cell lines are presented in Table II. Development of an overt and characteris-
tic cytopathogenic effect (CPE) certainly will provide an early indication of
viral contamination. However, the absence of CPE definitely does not indi-
cate that the culture is virus free. In fact, persistent latent infections may exist
in cell lines and remain undetected until the appropriate immunological, cy-
tological, ultrastructural, and/or biochemical tests are applied. Additional
host systems or manipulations, for example, treatment with halogenated nu-
cleosides, may be required for virus activation and isolation.
Screening techniques may be applied as an expedient compromise to
monitor for readily detectable viruses associated with cell lines. Egg inocula-
tions, in addition to selected co-cultivations and hemadsorption tests, can be
included, as can routine examinations for CPE using phase contrast micro-
scopy. Similar general tests are recommended by government agencies
when cell lines are to be used for biological production work. Detailed pro-
tocols have been provided elsewhere (Hay, 1992; Lubiniecki and May,
1985).
Despite such screens, latent viruses and viruses that do not produce overt
CPE or hemadsorption will escape detection. Some of these viruses could be
potentially dangerous for the cell culture technician. For example, Hantaan
6 Robert J. Hay
Table II
Representative Viruses of Special Concern in Cell
Production Work3
Host Virus
Human Human immunodeficiency viruses
Human T-cell leukemia viruses
Other endogenous retroviruses
Hepatitis viruses
Human herpes virus-6
Cytomegalovirus
Human papilloma virus
Epstein-Barr virus
Other Hantavirus
Lymphocytic choriomeningitis virus
Ectromelia virus
Murine hepatitis virus
Simian viruses
Sendai virus
Avian leukosis virus
Bovine viral diarrhea virus
a See Hayflick and Hennessen (1989) for more detail.
virus, the causative agent of Korean hemorrhagic fever, replicates in rat, cer-
tain human tumor, and other cell lines. Outbreaks of the disease in individ-
uals exposed to infected colonies of laboratory rats have been reported sep-
arately in five countries. An incident of transmission during passage of a cell
line was confirmed in Belgium. As a result of these findings, several rat and
rat/mouse hybrid cell lines expanded in this laboratory were screened using
an indirect immunofluorescent antibody assay (LeDuc et al., 1985) and were
found to be negative.
Concern over laboratory transmission of the human immunodeficiency vi-
ruses (HIV) also should be expressed. Several cases of probable infection
during processing in United States laboratories have been described, one
presumed to be caused by parenteral exposure and another by work with
highly concentrated preparations (Weiss et al., 1988). In this latter circum-
stance, strict adherence to Biosafety Level 3 containment practices is essen-
tial. More detailed discussion of safety precautions for work with human tis-
sues and cell lines is presented by Grizzle and Polt (1988) and by Caputo
(1988).
Other viruses that may present general problems in cell culture work in-
clude ectromelia virus, bovine viral diarrhea virus (BVDV), and Epstein-Barr
virus (EBV). Ectromelia virus, a member of the orthopoxvirus genus, is a natu-
ral pathogen of mice. Murine products such as ascitic fluids may transmit the