Cell Line Au thentication
This article provides a strategy and summarizes
steps for the authentication of cell line stocks. It serves
as a preface for the following article and for others in
this series offering detail on the characterization of
cells and cell lines. For those working with serially
propagated cells, it is absolutely critical that quality
control tests be applied periodically. Rationale and
pertinent key references are included here.
Literally hundreds of instances of cross-contamination
in cell culture systems have been documented
(Nelson-Rees, 1978: Nelson-Rees et al
., 1981.; Hukku et al.,
1984; MacLeod et al
., 1999). Many others have gone
unreported. 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 especially 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 are
certainly equivalent to many millions of dollars.
Accordingly, frequent reiteration of the details of
cell culture contaminations and of precautionary
steps to avoid and detect such problems clearly is
This article includes a review of quality control
steps applied to authenticate cell lines, i.e., to ensure
absence of microbial, viral, and cellular contamination,
as well as potential tests to verify the identity of
human cells. The approach suggested has been developed
during the establishment of a national cell repository.
Specific rationales for applying the tests indicated are included in this volume and are discussed
in more detail elsewhere (Hay et al
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 subsequent chapters of this series and
include, for example, phase-contrast and ultrastructural
morphologies; detailed cytogenetic analysis;
definition of protooncogene, oncogene, or oncogene
product presence, nature, and location; detailed evaluation
of intermediate-filament proteins; and demonstration
of tissue-specific antigens or production of
other specific products. These characterizations obviously
increase the value of each line for research and for
production work. However, cell resource organizations
need not repeat all of these tests before distributing the
stock cultures. Decisions must be made to establish the
most acceptable authentication steps, consistent with
maintaining the lowest possible cost, to provide a highquality
cell stock. Authentication can be considered the
act of confirming or verifying the identity and critical
feature 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 to identify or
reauthenticate that line specifically. Essential steps for
quality control will vary with the type of cell resource
constructed. Minimal descriptive data frequently 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 the production of vaccines or other biologicals. A
scheme illustrating the steps involved in developing
seed stocks is presented as Fig. 1.
Generally, starter cultures or ampules are obtained
from the originator, and progeny are propagated
according to the instructions to yield the first "token
freeze. Cultures derived from such token material are
then tested for bacterial, fungal, and mycoplasmal contamination.
The species of each cell line is verified.
These quality control steps are the minimum ones that
must be performed before eventual release of a line. If
these steps 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 distribution stock consists of ampules that are distributed
on request to investigators. The reference seed
stock, however, 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 most rigorous. The seed
stock here, like the master cell bank, is used as a reservoir
to replenish depleted distribution lots over the
years. By adherence to this principle, one can avoid
problems associated with genetic instability, cell line
selection, or transformation.
III. MICROBIAL CONTAMINATION
|FIGURE 1 Accessioning Scheme.
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, filamentous
fungi, and/or mycoplasma contaminants.
A. Bacteria and Fungi
Microscopic examination is not sufficient for the
detection of gross contaminations; even some of these
cannot be detected readily by simple observations.
Therefore, an extensive series of culture tests is also
required to provide reasonable assurance that a cell
line stock or medium is free of fungi and bacteria.
Details are given in the following article.
B. Mycoplasma Infection
Contamination of cell cultures by mycoplasma can
be a much more insidious problem than that created
by the growth of bacteria or fungi. Although the presence
of some mycoplasma species may be apparent
because of the degenerative effects induced, other
mycoplasma 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 to interpretation, if not negated in interpretation
entirely, when conducted with cell lines that
harbor mycoplasma. The seriousness of these problems
has been 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, M. salivarium, and M. fermentas)
are among those most frequently isolated. In the study
of Del Giudice and Gardella (1984) of the 34,697 lines
tested, 3955 (11%) were positive; 36% of these isolates
were mycoplasmas of human origin. A high incidence
of isolation of Mycoplasma hyorhinus
was noted that
may have resulted from using 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. Data showing that
these are not unusual findings are presented in Table
I. Results from seven laboratories published since 1980
indicate clearly that mycoplasma infection is still a
very major problem in the cell culture field. Some 5 to
20% of cultures tested were positive. This is the range
Protocols to test for mycoplasma infection are
included in the following chapter.
Four general recommendations can be offered to
avoid the problem. The implementation of an effective
regimen to monitor cell lines for mycoplasma is one
critical step. Quarantining all new, untested lines and
the use of 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 whenever a lapse in aseptic technique
occurs. When a primary tissue is used, e.g., a human
tumor sample, antibiotics may be employed initially,
but after the primary population has been grown out
and cyropreserved, reconstituted cells may be propagated
further in antibiotic-free medium.
Verification of the absence of viruses in cell lines is
recognized as a most significant problem. That these
may coexist as noncytopathic entities (e.g., the c-type
retroviruses) or in a latent form (e.g., papilloma viruses
and some herpes viruses) compounds difficulties in
detection. Judicious choices are necessary not only to
select appropriate methods available for recognizing
viruses associated with cell lines, but also to identify
the offending species. The nature of the cell resource,
its users, and budget available, plus the intended purposes
for which the lines will be needed, all affect decisions
on testing. More complete detail and protocols
are provided in the following article.
Wherever cells are grown in culture, serious risk
exists for the inadvertent addition and subsequent
overgrowth by cells of another individual or species.
One most certainly cannot rely on morphologic
criteria alone to recognize or identify cell lines. Datadocumenting
problems have been collected over the
years by groups offering identification services for
cell culture laboratories in the United States and
In one study, 466 lines from 62 laboratories were
examined. Of these, 75 (16%) were found to be identified
incorrectly. A total of 43 lines (9%) were not of the
species expected, whereas 32 lines (7%) were either
incorrect mixtures of two or more lines or were not the
individual line as stated (Nelson-Rees, 1978). Hukku et al
. (1984) examined 275 lines over a period of 18
months. Results of their analyses are summarized in
Table II. A total of 96 lines (35%) were not as indicated
by the donor laboratories. For purported human lines,
36% were not as expected, 25% were a different
species, and 11% a different human individual. More
recently, Drexler et al
. (1994), while developing a
resource of cell lines, reported that 10% of those
provided by outside investigators contained cells
different from those expected, probably due to
misidentification or cross-contamination.
To minimize the risk of cellular cross-contamination,
culture technicians require a laminar flow hood
for ideal operation. These individuals must be
instructed periodically to work with only one cell line
at any given time, use one reservoir of medium for
each line, and avoid introducing pipettes that have
been used to dispense or mix cells into any medium
Technicians must be reminded repeatedly to
legibly label each and every cell culture with designations,
passages, and dates. Labels of differing colors
can be used to more readily distinguish one cell line
from another during expansion.
Technicians must also be instructed to allow at least
5min of hood clearance time, with ultraviolet lights
and blower on, between cell lines when working on
more than one line during a particular period. The
inner surfaces of each hood should be swabbed with
70% ethanol between such uses.
The studies outlined earlier illustrate the severity
of the problem of cellular cross-contamination and
provide a strong rationale for vigilance in careful handling,
characterization, and authentication of cell lines.
A. Species Verification
Species of origin can be determined for cell lines by
a variety of immunological tests, by isoenzymology,
and/or by cytogenetics (Nelson-Rees, 1978; Hukku et al
., 1984; Hay et al
., 2000). The indirect fluorescent
antibody-staining technique is used in many laboratories
to verify the species of a cell line (for details,
see Hay et al
., 1992). Isozyme analyses performed on
homogenates of cell lines from over 25 species have
demonstrated the utility of these biochemical characteristics
for species verification (O'Brien et al
., 1977). By
determining the mobilities of three isozyme systems - glucose-6-phosphate dehydrogenase, lactic acid dehydrogenase,
and nucleoside phosphorylase - using
vertical starch gel electrophoresis, the species of origin
of cell lines can be identified with a high degree of certainty.
Alternatively, a standardized kit employing
agarose gels and stabilized reagents may be obtained
for this purpose (AuthentiKit, Innovative Chemistry,
Inc., Marshfield, MA).
Karyologic techniques have long been used informatively
to monitor for interspecies contamination
among cell lines. In many instances, the chromosomal
constitutions are so dramatically different that even
cursory microscopic observations are adequate. In
others, for example, in comparisons among cell lines
from closely related primates, careful evaluation of
banded preparations is required (Nelson-Rees, 1981;
Hukku et al.,
1984). Cytogenetics has the advantage of
detecting even very minor contaminants, on the order
of 1% or less in some circumstances. Furthermore, it
can provide precise identification of specific lines in
cases where marker chromosomes are known or
detected (e.g., Drexler et al.,
2002). However, cytogenetic
analyses are time-consuming and interpretation
requires a high degree of skill. The karyotype is constructed
by cutting chromosomes from a photomicrograph
and arranging them according to arm length,
position of centromere, presence of secondary constrictions,
and so forth. Automated analytical systems
are available but expensive. The "Atlas of Mammalian
Chromosomes" (Hsu and Benirschke, 1967-1975) illustrates
examples of conventionally stained preparations
from over 550 species. Detailed protocols are available
elsewhere (Hay et al.,
B. Intraspecies Cross-Contamination
With the dramatic increase in numbers of cell lines
being developed, especially from human tissues, the
risk of intraspecies cross-contamination rises proportionately.
The problem is especially acute in laboratories
requiring work with the many different cell lines
of human and murine origin available today.
Methods for verifying cell line species employing
enzyme mobility studies have been mentioned. Using
similar technology, but with different enzyme systems,
one can also screen for intraspecies cellular crosscontamination.
Cell lines from various individuals of
the same species often show different codominant
alleles for a given enzyme locus, the products of which
are polymorphic and electrophoretically resolvable. In
most cases, the phenotype for these allelic enzymes
(allozymes) is extremely stable. Consequently, when allozyme phenotypes are determined over a suitable
spectrum of loci, they can be used effectively to
provide an allozyme genetic signature for each line
under study (O'Brien et al., 1977; Hukku et al
The application of recombinant DNA technology
and cloned DNA probes to identify and quantitate
allelic polymorphisms provides additional powerful
means for cell line identification. These polymorphisms
can be recognized as extremely useful markers,
even if they are not expressed through transcription
and translation to yield structural enzymatically active
Hybridization probes to regions of the human
genome that are highly variable have been produced
for DNA profiling applications, including cell line individualization.
Profiles derived from human cell lines
can be interpreted best using scanning devices as the
patterns are complicated. For protocols and examples,
see Jeffreys et al
. (1985), Gilbert et al
. (1990), and Hay et al
. (1996, 2000).
Original procedures included fingerprints using
larger (10-20 bp) minisatellite DNA and Southern blotting.
With application of the polymerase chain reaction
(PCR), loci can be typed in hours rather than days.
Smaller microsatellite (2-6bp) loci have been identified
as well. Edwards et al
. (1992) demonstrated the
usefulness of these "short tandem repeat" (STR) loci in
recognizing individuals at the DNA level. One significant
advantage of STR loci over minisatellite repeats
is their small size. The small size of STR loci allows
multiplex PCR reactions to be developed in which
many loci are examined simultaneously in a single
reaction. Commercially available multiplexed STR
systems are available for routine screening of new cell
lines for authenticity as well as for validating any subsequent
distribution of replenishment cell lines. More
detail on STR profiling of human DNA cell lines is
available elsewhere (Hay et al
., 2000). A comprehensive
database can be accessed via the ATCC website at
http://www.atcc.org/cultures/str.cfm. STR loci are
among the most informative polymorphic markers
in the genome. The profiling process used at ATCC
involves simultaneous amplification of eight STR loci
and the amelogenin gene in a multiplex PCR reaction
(Promega PowerPlex 1.2 system). This allows for discrimination
of fewer than 1 in 108
amplicons are separated by electrophoresis and are
analyzed using Genotyper 2.0 software from Applied
Biosystems. Each peak in the resulting electropherogram
represents an allele that is alphanumerically
scored and entered into our database.
The classical method for intraspecies cell line individualization
involves karyotype analysis after treatment
with trypsin and Giemsa stain (Giemsa or G-banding). The banding patterns made apparent by
this technique are characteristic for each chromosome
pair and permit recognition, by an experienced cytogeneticist,
of even comparatively minor inversions,
deletions, or translocations. Many lines retain multiple
marker chromosomes, readily recognizable by this
method, that identify the cells specifically and positively
(Chen et al
. 1987; Drexler et al
. 2002). If readily
recognized marker chromosomes are present, contaminations
at less than 1% can be recognized with careful
scrutiny. This technique even permits discrimination
among lines from the same individual that cannot be
identified as different by DNA. These lines have
similar marker chromosomes, indicating a common
source, but each also has markers unique in type and
copy number. In contrast, Gilbert et al.
(1990) noted no
distinct differences among the nine HeLa derivatives
examined using the minisattelite probe 33.6 after Hae
III designation. Metabolic differences among the
HeLa derivatives (Nelson-Rees et al
., 1980) will ultimately
be traced to genetic differences among the
lines, as reflected by the unique cytogenetic profiles.
On this basis, the importance of documenting the
precise cytogenetics of cell lines used for production
purposes is very clear. Protocols are provided elsewhere
in this series.
V. ORIGIN AND FUNCTION
The markers used for verification of the source
tissues for cell lines are probably as numerous as the
types of metazoan cells. Major methods of demonstration
include an analysis of fine structure, immunological
tests for cytoskeletal and tissue-specific
proteins, and, of course, an extremely broad range of
biochemical tests for specific functional traits of tissue
cells. Ultrastructural features such as desmosomes or
Weibel-Palade bodies identify epithelia and endothelia,
respectively. The nature of intermediate-filament
proteins, demonstrated using monoclonal antibodies,
permits differentiation among epithelial subtypes,
mesenchymal, and neurological cells. Tissue- and
tumor-specific antigens can be used when reagents
are reliable and available. In addition, tissue-specific
biochemical reactions or syntheses may be used for
absolute identification if these features are retained by
the cell line in question. One excellent example is the
cell line NCI-H820 (ATCCmHTB 181) isolated from a
metastic lesion of a human papillary lung adenocarcinoma.
Cells of this line reportedly retain multilamellar
bodies suggestive of type 2 pneumocytes and
express the three surfactant-associated proteins SP-A (constitutively), SP-B, and SP-C (after dexamethasone
stimulation; A. Gazdar, personal communication). For
more examples, see other articles of this series and Hay
The overall utility of any bank of cultured cell lines
depends on the degree of characterization of the holdings
that has been performed by the originators, the
banking agency, and other individuals within the scientific
community. Ready availability at a reasonable
cost of the lines and such data, as well as the ability to
track distribution of the biologicals, are additional critical
considerations. Documenting the verification of
species and identity of each cell line, when possible, is
considered essential. Freedom from bacterial, fungal,
and mycoplasmal infection must be assured. However,
from the cell banking perspective, applying all possible
characterizations to every seed or master cell stock
developed is neither essential nor practical. At ATCC,
for example, screens for particular viruses have been
applied when a specific program support is available
for such testing. Similarly, the definition of ultrastructural
features, tumorigenicity, and functional traits,
for example, is performed with appropriate external
support and adequate rationale. The central responsibility
is to produce reference stocks, authenticated and
well characterized for multiple purposes, and to return
to those preparations over the years for the development
of working stocks for distribution or other
specific applications. Each replacement distribution
stock requires reauthentication prior to distribution
to intended users.
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