IJC
International Journal of Cancer

Check your cultures! A list of cross-contaminated or
misidentified cell lines
Amanda Capes-Davis1, George Theodosopoulos1, Isobel Atkin2, Hans G. Drexler3, Arihiro Kohara4, Roderick A.F. MacLeod3,
John R. Masters5, Yukio Nakamura6, Yvonne A. Reid7, Roger R. Reddel1 and R. Ian Freshney8
1

CellBank Australia – Children’s Medical Research Institute, Westmead, NSW, Australia
European Collection of Cell Cultures (ECACC) – Health Protection Agency, Porton Down, Salisbury, Wiltshire, United Kingdom
3
DSMZ – German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany
4
JCRB – Japanese Collection of Research Bioresources, Osaka, Japan
5
Institute of Urology, University College London, London, United Kingdom
6
RIKEN – BioResource Center Cell Engineering Division, Tsukuba, Japan
7
ATCC – American Type Culture Collections, Manassas, VA
8Centre for Oncology and Applied Pharmacology, Glasgow University, Glasgow, United Kingdom

Continuous cell lines consist of cultured cells derived from a specific donor and tissue of origin that have acquired the ability
to proliferate indefinitely. These cell lines are well-recognized models for the study of health and disease, particularly forcancer. However, there are cautions to be aware of when using continuous cell lines, including the possibility ofcontamination, in which a foreign cell line or microorganism is introduced without the handler’s knowledge. Crosscontamination, in which the contaminant is another cell line, was first recognized in the 1950s but, disturbingly, remains aserious issue today. Many cell lines become cross-contaminated early, so that subsequent experimental work has been
performed only on the contaminant, masquerading under a different name. What can be done in response—how can aresearcher know if their own cell lines are cross-contaminated? Two practical responses are suggested here. First, it isimportant to check the literature, looking for previous work on cross-contamination. Some reports may be difficult to find andto make these more accessible, we have compiled a list of known cross-contaminated cell lines. The list currently contains
360 cell lines, drawn from 68 references. Most contaminants arise within the same species, with HeLa still the mostfrequently encountered (29%, 106/360) among human cell lines, but interspecies contaminants account for a small but
substantial minority of cases (9%, 33/360). Second, even if there are no previous publications on cross-contamination forthat cell line, it is essential to check the sample itself by performing authentication testing.

Cell Lines as Model Systems

Key words: authentication, cell culture, cell lines, crosscontamination, DNA proﬁling, misidentiﬁcation
Additional Supporting Information may be found in the online
version of this article.
Novelty and Impact: This manuscript reviews the literature relatingto cross-contamination of cell lines. Its novelty comes from theinclusion of a list of known cross-contaminated cell lines (over 300
lines named), allowing researchers to check their own cell lines withreference to the article. Recent developments in this ﬁeld, including
methods of authentication testing, are also discussed.
Grant sponsor: National Health and Medical Research Council ofAustralia
DOI: 10.1002/ijc.25242
History: Received 24 Nov 2009; Accepted 18 Jan 2010; Online
8 Feb 2010
Correspondence to: Amanda Capes-Davis, CellBank Australia,
Children’s Medical Research Institute, Locked Bag 23,
Wentworthville, NSW 2145, Australia, Fax: þ61 2 9687 2120,
E-mail: acapdav@gmail.com

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Int. J. Cancer: 127, 1–8 (2010) V

Continuous cell lines represent a readily accessible and easily
studied resource for research into health and disease. Thesecell lines have acquired the ability to proliferate indeﬁnitely ifgrown in the appropriate culture conditions; usually this is arare event, since the majority of cells even in tumor tissue
will cease proliferation after a limited number of cell divisions.1 However, once established, a continuous cell line canbe repeatedly passaged, reliably recovers from cryopreservation and retains many of the properties of its cell type or tissue of origin.2,3 These advantages make continuous cell lines
effective, and widely used, model systems for normal cellular
processes and for a variety of disease states.
Cell lines are particularly attractive models for studying malignant disease. The genetic changes in tumor-derived cell lines
closely resemble those of the tumors of origin.4 Moreover, thegenetic changes required to establish continuous cell lines
from normal cells recapitulate many of the genetic changes
occurring in cancer.5,6 These genetic changes are required toovercome replicative senescence, in which normal cells continue to be metabolically active but are restricted from further
division.1 Cells able to overcome senescence continue

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proliferating until their telomeres become so short that thechromosomes undergo fusion-breakage-bridge cycles and theensuing genomic instability results in culture crisis. Occasionally (at a rate of 1 in 107 cells), an immortalized cell will
emerge from crisis and begin to divide again, yielding a continuous cell line.1 The changes seen throughout this process have
many parallels within cancer development, both for malignancy in general and when considering speciﬁc tumor types.7,8
Despite these advantages, numerous cautions have emerged
from the literature regarding appropriate use of cell lines asmodel systems.9,10 Even where cultures have been transformed
through the introduction of speciﬁc genes, cell lines that havepassed through replicative senescence and crisis are aneuploid,
heteroploid and genotypically and phenotypically unstable,
resulting in considerable heterogeneity within the culture.10
This instability will cause changes in the characteristics of thecell line but a further consequence may result: alterations in acell line can be accepted by the user as intrinsic to that culture
when there is actually extrinsic contamination present.

List of cross-contaminated cell lines

Figure 1. Rates of contamination for leukemia–lymphoma celllines. Percentages of cross-contaminated and Mycoplasmacontaminated cell lines from a dataset of 598 leukemia andlymphoma cell lines analyzed by the German cell line bank DSMZ.
‘‘False/authentic’’ refers to the presence or absence of crosscontamination; ‘‘mycoþ/myco ’’ refers to the presence or absenceof Mycoplasma contamination. Cell lines fall into the following
categories: authentic/myco(n ¼ 411, 69%); authentic/mycoþ

(n ¼ 108, 18%); false/myco (n ¼ 41, 7%) and false/mycoþ
(n ¼ 38, 6%). (Courtesy of Hans Drexler, DSMZ.)

Cell Line Cross-contamination and Misidentification
Cell lines become contaminated when a foreign cell line or
microorganism is introduced without the handler’s knowledge.
Although we do not wish to minimize the problem of microbial contamination, we will focus on cell line cross-contamination in this article. Cross-contamination may arise due to several causes, including poor technique (spread via aerosols or
accidental contact), use of unplugged pipets, sharing media
and reagents among cell lines and use of mitotically inactivated feeder layers or conditioned medium, which may carrycontaminating cells if not properly eliminated, for example, byfreeze-thaw and ﬁltration.11 In addition, a cell line can be
replaced by another as a result of misidentiﬁcation by confusing cultures during handling, mislabeling or poor freezer inventory control. Simple errors during labeling of culture ﬂasks,
truncation of the cell line name or typographic errors in a
published manuscript, can result in signiﬁcant confusion foryears after the event when another researcher attempts to use
the same cell line for ongoing experimental work.12
Cross-contamination may occur ‘‘early,’’ in which case theoriginal cell line has probably never existed independently, or‘‘late,’’ where the tested sample has been overgrown but otherstocks of the original may still exist.13 Unfortunately, celllines generally become cross-contaminated early, while still
within the originating laboratory.14 This is not surprising:
cultures can remain in crisis for a prolonged period of time
before emergence of an immortalized population and this is atime when a single cell, if introduced from a separate cellline, would rapidly take over the culture.
There are now a number of studies pointing out the severity of this problem and the need to take urgent action to minimize cross-contamination and its consequences.9,15–17 Ten
years ago, the German Collection of Microorganisms and Cell
Cultures (DSMZ) published data from its identiﬁcation testingof cancer cell lines submitted by various laboratories for de-posit at the cell bank.14 They found that 18% of 252 submitted
cell lines were cross-contaminated with more than half ofcases arising within only 6 laboratories. Subsequent work by
the DSMZ, extending the number of cell lines tested (Fig. 1),
shows that of 598 leukemia–lymphoma cell lines (the groupprovided with the most complete genetic data), 187 (31%)
were contaminated with Mycoplasma and/or a second cell linewith 38 (6%) of cell lines contaminated with both. These datasuggest that poor practice within some laboratories results incontamination of multiple cell lines with multiple contaminants, which can then be disseminated more widely if thesecultures are used by others.
Other studies have pointed out that testing of cell lines isoften infrequent, resulting in the failure to detect contaminatedsamples. John Ryan of Corning Life Sciences conducted surveys of seminar attendees in 1990, asking about Mycoplasma
contamination; 50% were not currently performing testing andonly 18% said they tested their cultures regularly. Almost 1 in
4 respondents (23%) had experienced Mycoplasma contamination, but with such a low level of testing, it is likely that thereal ﬁgure was much higher.18 Other data on cross-contamination were published in 2004 by researchers at the University ofCalifornia, Berkeley, where Walter Nelson-Rees worked on
this problem in the 1970s, focusing on the HeLa cell line.19 Of
483 respondents to a questionnaire on cell line usage, 35%
were using cell lines obtained from another laboratory rather
than a cell line repository, but almost half of all respondents
performed no testing for cross-contamination.20
A practical example of the consequences of cell line contamination can be found in a recent study published by Berglind
et al.21 The authors analyzed data within the UMD_p53 (2007)
database, which includes information on the p53 status of
1,211 cell lines. Discrepancies were found in p53 status for 23%(88/384) of cell lines where data have been published by 2
C 2010 UICC
Int. J. Cancer: 127, 1–8 (2010) Vindependent laboratories. It is likely that many of these discrepancies arose due to work with cross-contaminated samples; theauthors noted that many groups rely on previously publishedreports of a cell line’s p53 status,21 resulting in further confusion when interpreting results from these cell lines.
Cell banks have the expertise to detect such cross-contamination, and have been proactive in publishing reports of crosscontaminated cell lines,22,23 in publishing test results online24
and in developing new detection methods.25–27 Unfortunately,
however, cell banks have also reported reluctance from many
researchers to deposit cell lines for distribution.28 Such repositories specialize in the detection of cross-contamination and it
is unlikely that most laboratories have comparable resources inthis regard. In addition, many researchers obtain cell lines fromone another, rather than approaching the originator or purchasing the cell line from a cell bank performing quality control testing. This may be faster or cheaper than obtaining cultures from a reputable source but the practice makes
contamination more prevalent and harder to detect.

Practical Responses
Having deﬁned the problems, it is time to focus on what canbe done. Several cancer-related journals, including the International Journal of Cancer, have recently responded to theseissues by changing their policies to require evidence of authentication with all submitted manuscripts using continuous celllines.29,30 Their response underscores the need for laboratories
to come to grips with cell line cross-contamination and misidentiﬁcation. Every researcher involved in cell culture will
have cell lines currently in culture, stored in liquid nitrogen or
may be commencing work on a new cell line. Put practically,
how can you know if your cell lines are cross-contaminated?
There are 2 important answers to this question:
1. Check the literature, for example, by searching the
PubMed database using the cell line name and ‘‘crosscontamination.’’
2. Check your cultured cells. Unless a cell line has comedirectly from a repository or other laboratory performing identiﬁcation testing, it should be tested on arrival,
and all cultures should be periodically tested while inuse, before cryopreservation and when thawed from liquid nitrogen.31 A variety of methods are available forauthentication; for human cell lines, short tandem
repeat (STR) proﬁling is the current international reference standard and is recommended as an easy and economical way to conﬁrm cell line identity bycomparison to donor tissue or to other samples of thecell line held by laboratories worldwide.26

Checking the Literature: A List of
Cross-Contaminated Cell Lines
A 2004 survey of abstracts within the PubMed databasewould suggest that inappropriate usage of cross-contaminatedC 2010 UICC
Int. J. Cancer: 127, 1–8 (2010) V

3cell lines is increasing,20 despite many years of publication on
this issue. It is possible that many researchers simply cannot
ﬁnd existing references to cross-contamination so, to makethis already published work more accessible, we have surveyed the literature and other online resources for references
to cell line contamination. The resulting list of cross-contaminated cell lines is included as Electronic Supporting
Information.
To generate this list, the authors examined the PubMed
database, references within other articles relating to this topic
and the websites of 5 cell banks: the American Type Culture
Collection (ATCC), DSMZ, European Collection of Cell Cultures (ECACC), Japanese Collection of Research Bioresources
and the RIKEN Bioresource Center Cell Bank. A Wikipedia
list of contaminated cell lines was also accessed (http://en.wikipedia.org/wiki/List_of_contaminated_cell_lines). Cross-contaminated cell lines are listed by name along with their species and cell type (both claimed and actual), the name of thecontaminating cell line where identiﬁed, the reference inwhich this was reported and the PubMed ID number whereavailable. Notes are also included for some cell lines. The list
is made available in Excel spreadsheet or PDF format foreasy accessibility.
The cell lines listed within this database are divided into 2
tables. Supporting Information Table 1 contains those celllines where cross-contamination occurred as an early event,and thus where there is no original material remaining. Supporting Information Table 2 contains those cell lines where itis thought cross-contamination occurred as a late event andwhere original stocks may still exist. A full list of referencesis also given.
The current list of cross-contaminated cell lines (version
6.4) contains 360 cell lines, 346 in Supporting Information
Table 1 and 14 in Supporting Information Table 2, drawn
from 68 references. Cell lines affected are primarily human,although cultures from at least 8 other species are included,and come from a wide spectrum of tissue types. The cell ortumor type is given within the list where known; extensive
work has been done by some cell banks and laboratories inthis area to characterize the actual cell type or tumor
type.22,32 In some cases, this work has shown that a cell line
carries the correct name but its cell or tumor type has been
incorrectly identiﬁed, for example, the cell line RPMI-6666
was initially thought to have come from Hodgkin lymphoma
but is now known to be an EBV-positive B-lymphoblastoid
cell line.22
Common features for cross-contaminating cell lines within
the current list are summarized in Table 1. It can be seenthat most cross-contamination events have arisen fromwithin the same species but a substantial minority (9%, 33/360) involved cross-contamination from a second species.
For the intraspecies contaminants, all of those detected were
human but it is likely that this relates to the difﬁculty ofdetecting intraspecies contaminants for nonhuman species.
The commonest contaminant remains the HeLa cell line

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List of cross-contaminated cell lines

Table 1. Cross-contaminating cell lines
Type of contaminant

Number of celllines affected

Intraspecies

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Human

324

Table 2. Websites for ongoing access to the list of
cross-contaminated cell lines
Cell bankWebsite address

ATCC

http://www.atcc.org/

CellBank Australia

http://www.cellbankaustralia.com/

Nonhuman0

DSMZ

http://www.dsmz.de/Interspecies

33

ECACC

Correct name—incorrect cell
type (misidentiﬁed)1

3

http://www.hpacultures.org.uk/collections/ecacc.jsp

Total

360

Contaminating cell line—12
most frequent

Number of celllines affected

HeLa (human cervical adenocarcinoma)

106

JCRB

http://cellbank.nibio.go.jp/

RIKEN Bioresource
Center Cell Bank

http://www.brc.riken.go.jp/lab/cell/english/
guide.shtml

T-24 (human bladder carcinoma)

18

Checking Your Cultures: Authentication of Cell Lines

HT-29 (human colon carcinoma)

12

CCRF-CEM (human acutelymphoblastic leukemia)

9

Even if a search of the literature shows no indication that acell line is contaminated, it is still essential to test the sample
that you are working with. Authentication testing should beconsidered in a positive light, as an essential part of good cell
culture practice33 and as an assurance for researchers, funding bodies and journals that the cell line used is a valid experimental model.17
There are a number of methods for testing cell line identity. When the issue of cross-contamination was ﬁrst identiﬁed, HeLa contaminants were detected through a combination of isoenzyme and chromosomal analysis.19,34 Bothtechniques continue to be used but there are also many
newer molecular approaches. Commonly used authentication
methods are summarized in Table 3; what factors should beconsidered when choosing between these methods?
The expertise of the laboratory holding the cell line is an
important factor. For example, laboratories with experiencein cytogenetics would have the skills to identify species
through karyotype analysis and cell lines through the presence or absence of appropriate markers.35 Although this is anolder approach, it still allows clear identiﬁcation of cell lines,
and many cell banks have published karyotypic informationon their cell lines to allow comparison to well-characterizedstocks. It should be noted that tumor-derived cell lines canbe surprisingly difﬁcult to harvest for cytogenetic analysis35
and are typically heteroploid making interpretation difﬁcult:the experience of the operator is important for success.
The species of cell lines held within the laboratory is also
important. Although some authentication methods can beused on more than 1 species, molecular methods such as
STR proﬁling are only successful for a single species; otherspecies will simply fail to amplify.26 This may not be an issue
for laboratories working only with human samples but clearly
is a signiﬁcant factor for groups working with rodent celllines. In this regard, multilocus DNA ﬁngerprint analysis hasa clear advantage, since probes are able to hybridize to awide variety of species.25 Unfortunately, although successful
within a single laboratory, it can be challenging to compare
DNA ﬁngerprints across several experimental runs, and it is
difﬁcult to exchange data among laboratories or for cell

K-562 (human chronic myeloid leukemia)

9U-937 (human lymphoma)

8

OCI/AML2 (human acute myeloid leukemia)8

Hcu-10 (human esophageal carcinoma)27

M14 (human melanoma)

7

HL-60 (human acute myeloid leukemia)

6

PC3 (human prostate carcinoma)

6

SW-480, SW620 (human colon carcinoma)36

1
For additional misidentiﬁed cell lines see Drexler et al.22 2Hcu-10
carries the same genetic identity as Hcu-18, Hcu-22, Hcu-27, Hcu-33,
Hcu-37 and Hcu-39; it is unclear which is the correct identity (seeElectronic Supporting Information for reference). 3SW480 and SW620
come from the same donor and therefore carry the same genetic
identity (see Electronic Supporting Information for reference).

(29%, 106/360), followed by T-24 (5%, 18/360) and HT-29(3%, 12/360).
It is important for such a list to be continually updated
and feedback is welcome for this purpose. An earlier versionof the database was released online by ECACC31; 6 cell banks
have now agreed to make the database available online andto update this information where necessary. Current website
addresses for access to the list of cross-contaminated celllines are given in Table 2. In future, it is envisaged that thecurrent list of misidentiﬁed cell lines will be included in a
new initiative improving access to authentication data. The
Standard Development Organization at the ATCC is in theprocess of producing an international standard for humancell line identiﬁcation based on STR proﬁling (ATCC SDO
Workgroup ASN-0002, manuscript submitted). Strict criteria
for STR proﬁles derived from cancer cell lines are being
developed. One consequence of this initiative is that funding
is being sought for a quality controlled and curated cell line
database with free access into which the database describedhere will be incorporated.

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Name

Description

PurposeReferences

Chromosomal analysis/karyotyping

Involves preparation of a metaphase spread with chromosome banding and painting to identify chromosome
number and markers

Separates species, plus
individual cell lines if
detailed analysis
performed

Ref. 35

Isoenzyme analysis

Biochemical method separating isoenzymes by electro- Separates species, somephoresis; isoenzyme mobility may vary within or across times individuals
species. Kits available include the Authentikit gel electrophoresis system

Refs. 36,37

Multilocus DNA ﬁngerprint analysis

Molecular method detecting variation in length within
minisatellite DNA containing variable numbers of tandem repeat sequences. Analysis is by Southern blot
hybridization using probes 33.6 and 33.15, M13
phage DNA, or oligonucleotide sequence

Separates individual celllines across multiple
species

Refs. 25,38

Short tandem repeat (STR) proﬁling

Molecular method detecting variation in length within
microsatellite DNA containing variable numbers ofshort tandem repeat sequences. Analysis is by PCR
with comparison to set size standards; usually available in a kit format allowing ampliﬁcation of up to 16
loci

Separates individual celllines within a single
species

Refs. 26,39

Polymerase chain reaction (PCR)
fragment analysis

Molecular method involving ampliﬁcation of speciﬁc
genes or gene families, aiming to detect variations in
exon/intron sequence, transcript splicing, or the presence of pseudogenes. Genes examined include the aldolase gene family and the beta-globin gene

Separates species only

Refs. 40,41

Sequencing of ‘‘DNA barcode’’ regions Involves sequencing of a DNA fragment from the mitochondrial gene cytochrome c oxidase subunit I, withcomparison to sequence obtained from online databases. This ‘‘DNA barcode’’ has been shown in practice to distinguish a broad range of animal species

Separates species only

Refs. 27,42

banks to publish such ﬁngerprints online. It is advisable toalways compare the test sample to a known sample within
the same experiment, ideally using DNA from the blood ortissue of the original donor.
The obvious advantage of STR proﬁling lies in the use ofcontrol samples to generate a numerical code for each sample, which precisely identiﬁes that cell line and which can bereadily shared and published online. It is primarily for thisreason that STR proﬁling is recommended as an internationalreference standard for human cell lines26 and accepted within
the legal system for human identity testing.39 STR proﬁling isbased on the presence of STRs within the human genomethat exist at variable lengths throughout the population. Eachof the repeat regions to be analyzed (usually tetra or pentanucleotide repeats in noncoding sequence) is ampliﬁed by
PCR using primers carrying ﬂuorescent tags and electrophoresed in a sequencing gel; the precise length of each allele isdetermined and compared with size standards and controls.
This allows identiﬁcation software to assign a number toeach allele at that locus (see, e.g., Fig. 2). The combination ofmultiple loci—classically 13, as used in the FBI Laboratory’s
Combined DNA Index System (CODIS)—gives sufﬁcient
data to uniquely identify that individual.
STR proﬁles for individual cell lines and panels have now
been reported by many laboratories (e.g., Ref. 44) and are
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published online by several cell banks. However, there aresome cautions to be aware of when using this approach. It is
accepted within the forensic ﬁeld that tumor samples are notas genetically stable as other tissue sources for STR proﬁling,
because of loss of heterozygosity and microsatellite instability.45,46 This is even more evident in tumor-derived cell lines,
where evolution or genetic drift continues to occur with passage.47 When searching an online database of STR proﬁlesfrom cell lines, the user needs to look for close matches andnot just identical matches; most studies would agree that80% similarity is an appropriate threshold for declaring amatch when comparing cell line proﬁles.26,44 There may also
be a signiﬁcant start-up cost if testing in-house; in addition
to an STR kit, access to methods for DNA extraction, precise
quantitation, fragment analysis and software for STR proﬁleidentiﬁcation is required.
The fact that STR proﬁling is only suitable for distinguishing cell lines of a single species has led to the need to
re-examine authentication of nonhuman cell lines. Laboratory rodent samples will always be difﬁcult to identify precisely due to inbreeding; laboratories working with rat or
mouse cultures may wish to examine strain identity rather
than authentication of individual cell lines, particularly ifthey have expertise in single nucleotide polymorphism (SNP)or single sequence length polymorphism (SSLP) analysis,

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Table 3. Commonly used methods for authenticating cell lines

List of cross-contaminated cell lines

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Figure 2. Example of STR proﬁle generation and interpretation. An example of STR proﬁling is given for the JFCF-6 cell ﬁbroblast strain and
3 of its immortalized derivatives, JFCF-6/T1.D, JFCF-6/T1.J/1.3C and JFCF-6/T1.Q.43 Derivatives were established after transfection with SV40
early region DNA and were handled by CellBank Australia through its Culture and Return service. DNA from each culture was ampliﬁedusing the AmpFlSTR Identiﬁler PCR Ampliﬁcation Kit (Applied Biosystems, Mulgrave, Australia), which includes primers for 16 STR loci.
Ampliﬁed sequence was analyzed using an ABI PRISM 3100 Genetic Analyzer and data ﬁles were assessed using GeneMapper ID software(Applied Biosystems). (a) Photographs taken of each culture, comparing parental cells to the morphology of each derived cell line (scalebar ¼ 100 lm). Each derivative has a markedly different morphology, showing the need for authentication testing to conﬁrm thatderivatives correspond to the parental strain. (b) Examples of STR peak ampliﬁcation for the D16S539 locus of each culture. Ampliﬁcation
varies at this locus due to genetic drift during establishment of the 3 JFCF-6–derived cell lines. The peaks shown correspond to speciﬁcallele sizes known to exist at this locus and conﬁrmed using size standards and controls supplied with the kit (data not shown). (c). STR
proﬁles for JFCF-6 and derived cell lines; the locus shown in B, D16S539, is highlighted in grey. Despite the differences seen due togenetic drift, the proﬁles for derived lines closely match the parental cell strain and all of these cultures are correctly identiﬁed.

which can be used for strain identiﬁcation.48,49 SNP analysis
can also be used to identify individual samples50 and has
been used for cell line authentication,51 making it a methodof great promise for application to human and nonhumansamples alike. Laboratories working on speciﬁc cell types
may be able to use expressed markers for identiﬁcation, as 1laboratory has done recently, publishing a technique for
identiﬁcation of hybridomas based on sequencing of lightchain variable regions.52
A simple method has recently emerged to help detect interspecies contamination. The term DNA barcoding here refers

to amplifying a speciﬁc 648 bp fragment of the mitochondrial
gene, cytochrome C oxidase subunit I (COI), using primersdeveloped by Folmer et al.53 Sequence divergences within thisfragment allow species discrimination across almost all animal
phyla.42 Although debate is ongoing as to whether DNA barcoding is sufﬁcient for assignment of species in taxonomic
terms,54 it is clear that the technique can readily identify the
species of an unknown specimen if compared with previously
sequenced reference material in online databases.55 DNA barcoding has been tested for species identiﬁcation of cell lines27
and its use would reduce the incidence of interspecies cell line
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Conclusions
Cell line contamination is a serious issue that detracts fromthe use of cell lines as model systems to help us understand abroad range of diseases, including cancer. Responding practi-

cally by checking each cell line before it is used, searching forprevious references and authenticating the sample itself isworthwhile and will reduce the risk and subsequent consequences of contamination long-term.

Acknowledgements
The authors gratefully acknowledge the work of many cell banks andlaboratories working in this area, and those responsible for compilingthe list in Wikipedia, and regret that there is insufﬁcient space toinclude all references here. A complete list of publications on cross-contamination can be found in the Electronic Supporting Information. Elsa
Moy is thanked for her work in handling the cell lines shown in Figure
2. CellBank Australia was established by a joint venture of the Children’s Medical Research Institute, Cure Cancer Australia Foundation andNational Breast Cancer Foundation, and by an Enabling Grant of the
National Health and Medical Research Council of Australia.

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Mini Review

contamination, found here to cause almost 1 in 10 of all published cross-contamination events.
Whatever the authentication method used, it should beclearly recorded within the researcher’s experimental notes,
and the result should be linked if possible to the laboratory’s
liquid nitrogen records, so that quality control for frozen
vials is clearly evident. When publishing experimental work,
the Material and Methods section should include the correct
and full name of the cell line used, its origin (with appropriate references), the source of the cultures used and details ofauthentication testing.

Mini Review

8

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