November 15, 2014 (Vol. 34, No. 20)

Angelo DePalma Ph.D. Writer GEN

Don’t fall into the cell line misidentification trap. Authenticate! What you don’t know—or what you refuse to acknowledge—will hurt you.

Calls for diligence in cell line authentication must be issued with greater urgency, insist scientific stakeholders on reproducibility. According to one such stakeholder, Maryellen deMars, Ph.D., senior director of the standards resource center at American Type Culture Collection (ATCC), “The incidence of incorrect cell line use may be as high as 35%.”

As a consequence, funding agencies and publications increasingly demand that researchers authenticate cell lines before undertaking long projects or submitting papers.

In 2012 the ATCC Standards Development Organization published ASN-0002: Authentication of Human Cell Lines: Standardization of STR Profiling, the current consensus standard on short tandem repeat (STR) analysis, which is available for download through the ATCC website.

ATTC has replaced isoenzymology with the cytochrome C oxidase I (CO1) test for species-level authentication. CO1 is a mitochondrial gene across diverse phyla in the animal kingdom. Because the gene is not well conserved between species, it functions as a genetic species marker. ATCC scientists have developed a PCR-based CO1 speciation assay by designing primer pairs that recognize specific species uniquely. The test distinguishes from among cell lines arising from pig, human, cat, Chinese hamster, Rhesus monkey, sheep, horse, African green monkey, rat, dog, mouse, rabbit, goat, and cow.

ATCC’s standards committee is working on additional authentication methods to supplement STR profiling and CO1 testing. The organization uses both STR and CO1 in its internal quality programs, but offers only STR as a service. “We’re looking into providing CO1 at some future date,” Dr. deMars says.

PCR-based authentication is based on analysis of genes that are not well conserved between species or individuals within species. The size of amplicons in CO1, for example, is definitive for species. Amplicon differences in STR human genome analysis digs deeper, distinguishing one individual within a species from another. According to ATCC, at least eight loci are required to positively identify a human cell line through STR; some authentication service providers use more than eight.

Combining species and subspecies tests provides verification of species and contamination, according to Dr. deMars: “CO1 broadens our ability to look at misidentification. Now you can detect if other species exist in a cell line.”


LNCaP cells showing staining of androgen receptor (red) and beta-catenin (green) by immunofluorescent techniques. The LNCaP line was originally established from a metastatic lesion of human prostatic adenocarcinoma. This image, provided by Sanford Research, shows cells from the LNCaP clone FGC (ATCC® CRL-1740™). [Vasudha Sundram]

How Deeply?

Analysis depth is a common question arising in cell line authentication. “If a customer only needs species-level authentication, which is quite common, an isoenzyme test will do,” notes Deborah Lee Letham, Ph.D., senior scientist, methods development at Charles River Laboratories. “But if they’re using a recombinant cell line, then they need to look for the recombinant gene at the subspecies level.”

Species-level authentication distinguishes between, say, cells arising from human or mouse; subspecies analysis differentiates between individuals of the same species.

For human cell line validation, STR is the preferred analysis method. Various groups are developing STRs for other lineages as well, for example, Vero cells and mouse cell lines.

PCR-based STR profiling helps detect cell lines that have been misidentified, cross-contaminated, or that have drifted genetically. STR will flag human cell lines that have been contaminated with HeLa cells, for example. “Contaminating cells may take over old cells, to the point where no original cells remain,” Dr. Letham says. An STR report from a reputable service organization meets authentication requirements of funding agencies, publications, and quality control.

Lab managers should consider characterizing cell lines extensively as soon as they acquire the cells, then use a preserved sample or DNA extract from that line to authenticate subsequent generations. Cells obtained from reputable vendors or organizations are preauthenticated. Purchasers should in any case request proof of authentication before buying.

“A top-level analysis might be sufficient, but if you have the resources, it sometimes makes sense to go more deeply, to strain identification, which gives you more information about your line,” Dr. Letham advises.

Unless they are fortunate enough to have a genetics core facility at their disposal with experience running authentication protocols, labs would do well to outsource these services. The tests require the appropriate positive and negative controls and adherence to protocols and strict quality practices.

Top of the List

Unless labs acquire cell lines from a reputable commercial source, authentication should be near the top of the to-do list for any group using cells for research, development, and protein production.

According to Gabriela Saldanha, strategic marketing manager at Promega, the biggest issues today in cell line authentication are a lack of awareness of the problem and cell biologists’ lack of familiarity with authentication technology. “They’re first and foremost cell biologists, not molecular biologists,” Saldanha explains. “The two disciplines are very different worlds.”

Furthermore, even biologists who have become aware of authentication issues through conferences and articles, and who understand its rationale, probably lack the requisite equipment. “Confusion exists about how you even get started.”

Authentication issues hit hardest when scientists generate data and publish results while under the misapprehension that their cells are of a particular line, when in fact their cells have been infiltrated, or completely overcome, by cells of a different line. As many as 25% of all manuscripts employing human cell lines may not have actually used lines they list in the methods section. Patents have similarly been issued, and clinical trials initiated, on the basis of results from the wrong cell line.

HeLa cells are particularly problematic because of their ubiquity in cell biology laboratories and their tendency to overgrow most other research cells. Senior-level scientists will take precautions to prevent a few HeLa cells from slipping into a different human-derived culture, but undergraduates and graduate students who put a premium on finishing an experiment or a degree requirement may cut corners.

“Mixed cultures are actually quite rare,” Saldanha tells GEN. “The fastest-growing cells will overcome cultures. You may have been working with gastrointestinal cells at the beginning of a project, but two years later, when you’re getting ready to publish, you may be working with only HeLa cells.”

Following Best Practices

Cell line problems arise when labs neglect best practices for cell line authentication. Labs, however, might easily take preventative measures. One such measure, Saldanha notes, would involve taking cell line aliquots and storing them in a freezer. If identity or contamination were to become an issue later on, labs could confirm the identity of the cells in current use against reference samples. Labs could also run checks at the end of a project, before publication or subsequent utilization of their data.

Problems also arise when principal investigators obtain cells from colleagues who have not made the effort to authenticate, or if they work with multiple cell lines. “There’s a potential for things to get mixed up, mislabeled, especially when you work with multiple cells in the same hood,” Saldanha warns.

And unfortunately many researchers, primarily academics, are too focused on costs. “They think, ‘It will cost me more money to do this,’ without considering the time, money, and effort that could be wasted if they were to start a long-term study with the wrong cell line.”

Mind-Boggling Lapses

“With the great importance placed on cell lines in research, the mind boggles why cell line misidentification is still a talking point,” marvels Michael Ojo, a cell line authentication specialist at LGC Standards.

Scientists have a multitude of resources available, many of them free, from the ever-growing misidentification cell line list to public cell line reference databases offered by ATCC—not to mention the ATCC Standards Development Organization document, ASN-0002, referred to earlier. “This document,” Ojo says, “outlines a working standard which can be utilized and implemented in any lab.”

If this is too time consuming for the researcher, there are also authentication services, which for a reasonable fee provide confidence that lines are genuine. Customers generally receive results within one week.

Authentication by STR profiling has become the standard for authenticating human cell lines and will remain so for the foreseeable future due to the technique’s effectiveness and the existence of comprehensive reference databases. However, Ojo does not see a parallel effort for authenticating popular animal cell lines.

“The currently available technique, sequencing either cytochrome B or cytochrome C—also known as DNA barcoding—while adequate, does not offer the same resolution as STR profiling for humans. While we also offer an animal authentication service based on these methods, we are always on the lookout for the next move,” continues Ojo. “I expect, with our strong research division, that we will be at the forefront of bringing a new offering to the market to put the authentication of both human and animal cell lines on an even keel.”

Misidentified cell lines will continue to cast a shadow over biology and medicine not because authentication technology does not exist or is too costly, but because of human frailty. “Ignorance can no longer be used as a defense because cell line authentication is not a new issue,” insists Ojo.

Although ignorance may be indefensible, it persists, as does wishful thinking. Such lapses contribute to a general unwillingness to take the precaution of authentication. The problem, in a word, is complacency.

Ojo muses that complacency would fade if demands for authentication, already instituted by high-impact journals and prominent funding bodies, were to become all but universal. “But, really,” he asks, “shouldn’t a scientist want to ensure their work is accurate and reproducible without being pushed?”

“Awareness of the importance of cell line authentication is there, but it is not as ingrained as it should be among biologists,” reflects Dr. deMars of ATCC. “We hope that development of consensus standards, and support by funding agencies and journals, will put external pressure on biologists and that authentication will become more routine.”


To help clients achieve the routine detection of misidentified or contaminated cell lines, LGC Standards maintains a cell line authentication program. Under this program, the company’s specialists perform PowerPlex 16HS short tandem repeat analysis, compare the results against its database to identify the cell line, and generate a comprehensive report. [Andrew Brookes]

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