November 15, 2017 (Vol. 37, No. 20)

Researchers May Be Slow to Implement Protocols Unless They Are Pressured

Imagine publishing a paper, accepting a research grant, or recruiting patients for a clinical trial only to discover that the esophageal cancer cell line used in your disease model actually came from a different tumor type. It’s a scientist’s nightmare, but it became an unfortunate reality for researchers working with the widely used esophageal cancer cell lines, SEG-1, BIC-1, and SK-GT-5, when an alarming report in the Journal of the National Cancer Institute, published in 2010, ripped away the mask concealing these misidentified cell lines.1

The really frightening part, however, is that this example does not represent a single, isolated event, but rather, an insidious recurring issue that has affected the entire biomedical research community and has even received attention from public media outlets. In 2014, NPR broadcasted a report about a misidentified melanoma cell line, MDA-MB-435, that researchers have used for years as a model for human breast cancer.2 Now the validity of all that data, which included over a thousand publications, comes into question.

In addition to littering scientific literature with potentially erroneous conclusions, research studies using contaminated or misidentified cell lines receive an estimated $700 million dollars in grant funding every year.3 To break this cycle, the National Institutes of Health and many scientific journals have revised their submission guidelines for research grants and peer-reviewed articles to include requirements for authenticating cell lines. The effectiveness of these new policies may depend on how these organizations decide to enforce them, but they represent a much-needed step toward solving a problem that has plagued the scientific community for over 50 years.

Short-Tandem Repeat Analysis: A Scientist’s Insurance Policy

Well-established technologies for authenticating human cell lines exist, but barriers to their use—including awareness, complacency, time, and cost—have frustrated efforts to get past the current cellular identity crisis. Cell line authentication advocate and global strategic marketing manager at Promega, Gabriela Saldanha, Ph.D., offers her perspective to researchers who might list cost as a reason to skip an identity check: “[Cell line authentication] is like an insurance policy. Nobody likes paying for insurance, but you never know when you’re going to need it.” Unfortunately, many biomedical researchers do not realize or acknowledge that they’re working in a flood zone—where estimates for the total percentage of cell lines that have been contaminated or misidentified range from 18% to 36%.

The current gold standard for authenticating human cell lines, short-tandem repeat (STR) analysis, is an identification technique used by forensics labs during criminal investigations. Only 2–5 base pairs in length, STRs are short, repeated sequences in DNA that provide a powerful tool for human identification, because the number of repeated units can vary significantly between individuals. The location on a chromosome where an STR occurs is known as an STR locus, and each locus represents a different contour in an individual’s genetic fingerprint.

Do-it-yourself types can purchase STR assay kits designed for cell line authentication for less than $14 per test from companies such as Promega and Thermo Fisher Scientific. These companies have optimized fluorescently labeled primers to allow co-amplification of multiple STR loci in a single PCR reaction (Figure 1).

Figure 1. Promega’s GenePrint 10 System and GenePrint 24 System are optimized for amplification of purified DNA or direct amplification of DNA from storage card punches using the GeneAmp PCR System 9700 thermal cycler. Either approach may be used to identify STR loci, which consist of repetitive DNA sequences with varying numbers of repeats. Each STR locus can be amplified by PCR, and the amplified products can be labeled with different fluorophores, making the products easy to distinguish by size and color.

For example, Promega’s GenePrint® 10 and GenePrint® 24 Systems co-amplify amelogenin, a marker that indicates an individual’s sex, plus 9 and 23 autosomal STR loci, respectively. After amplification, researchers can perform size- and color-based separation and detection of STR loci using capillary electrophoresis. Finally, they can generate an STR profile and compare it to a reference to verify the identity of their cell lines.

Some researchers may lack experience with the molecular biology techniques needed for STR, or they may be unable to access the right equipment. These researchers may resort to sending samples to a service provider, such as Genetica (Figure 2), ATCC, or Multiplexion. Although scientists have cited cost, time, and delays in research as the top three barriers to cell line authentication,4 these companies have worked hard to establish fast, affordable services.

“[Genetica] offers several different STR report options, from basic Excel spreadsheets to formal comparative reports, to fit the budget of any research lab,” said Erin Hall, director of cell line authentication services at Genetica, and customers can expect to receive their results in as few as two business days. Genetica’s standard cell line authentication service uses amelogenin to distinguish between cell lines derived from male and female individuals plus 15 STR loci to validate cell lines (Figure 2), and they recently added a marker to detect mouse DNA contamination.

“The test can easily pick up a low level of mouse [DNA] contamination in a human specimen,” said Hall, who explained that “this additional marker is especially helpful for researchers using mouse xenograft models of human tumors or those that are using both human and mouse cell lines simultaneously.”

Figure 2. An electropherogram of PowerPlex®16HS results, provided by Genetica, showing a mixture of cell line MDA-MB-231 (breast adenocarcinoma) and cell line A549 (lung carcinoma). The presence of more than two peaks at more than one genetic marker is a good indicator that a mixture may be present; the other potential cause of additional peaks is genetic instability, an occurrence that can be normal for certain cancer cell lines. In this example, note the presence of the “Y” chromosome (amelogenin marker; bottom panel); A549 is derived from a male individual, whereas MDA-MB-231 is derived from a female. This helps lead to the conclusion that a mixture is present, as opposed to genetic instability.

Of Mice and Men: Authenticating Non-Human Cell Lines

A significant number of scientific studies use non-human cells, which are just as susceptible to cross-contamination, misidentification, and mislabeling as those from human samples. Although Genetica’s mouse marker can help identify cross-contamination between mouse and human cell cultures, a major limitation of STR analysis is that it’s currently only available for human cell line authentication.

In an effort to build the infrastructure needed to enable the identification of non-human cell lines using STR analysis, a number of organizations, including Genetica and ATCC, have joined the National Institute of Standards and Technology to form the Mouse Cell Line Authentication Consortium (MCLAC). “Mouse is the next most popular cell line after humans, so it’s the logical extension of bringing STR technology to another species,” said Maryellen de Mars, Ph.D., vice president, Standards Resource Center, ATCC.

In 2012, ATCC’s Standards Development Organization released a consensus standard for authenticating human cell lines using STR analysis. In addition to identifying amelogenin and 8 autosomal STR loci as markers for authentication, the standard also recommends a minimum matching criterion of 80% to determine if two cell lines are really the same.

According to Dr. de Mars, it will “take time and effort” to develop a similar standard for mouse cell line authentication: “[MCLAC] needs to identify the correct combination of loci needed to sufficiently distinguish mouse cell lines using STR analysis.”

Since offspring can inherit specific STR polymorphisms from their parents, the substantial inbreeding of mice by the scientific community means that researchers will need more markers to distinguish between cell lines: thus far MCLAC has established a combination of 19 mouse STR loci and 2 human markers. In addition to identifying robust STR markers, MCLAC will also need to generate a mouse cell line database, establish an algorithm for comparing STR profiles, and define matching criterion.

In the meantime, ATCC offers a mitochondrial cytochrome c oxidase subunit 1 (CO1) assay that uses DNA barcoding to discriminate between species, which researchers can use both to confirm their cell lines at the species level and to detect interspecies contamination (Figure 3). “Authentication actually involves both identity and purity,” explained Dr. de Mars. In addition to identifying the cell line, authentication should test for cross-contamination between cell lines, as well as viral and mycoplasma contamination.

Figure 3. The aggressive HeLa cell line, shown in this fluorescent microscopy image with cytoskeletal (magenta) and nuclear (cyan) components highlighted, looks similar to other cell lines, but it’s one of the most common contaminants identified in ICLAC’s Register of Misidentified Cell Lines. To confirm the identity of its cell lines, ATCC has used DNA polymorphisms in addition to enzyme polymorphisms, HLA typing, and karyotyping. Such work may require STR profiles, which are available for all ATCC human cell lines.

Catching the Genetic Drift (Evolution in a Petri Dish)

Although cell culturists typically think of contamination in terms of microorganisms—bacteria, fungi, viruses, and protozoa—cross-contamination between cell lines poses a particularly worrisome threat. Contamination events that introduce stray cells from another culture can go unnoticed. And unnoticed problems can multiply.

As Steve Jackson, Ph.D., application scientist at Thermo Fisher Scientific remarked, “If something has a growth advantage, even a small contaminant is going to overgrow the cell line in a very short time.” Nothing exemplifies this truth more than the HeLa cell line, which the International Cell Line Authentication Committee (ICLAC) has identified as a contaminant in 113 different cell lines (Figure 3).

Not only can natural selection lead to a hostile takeover of your culture by HeLa cells, but it can also lead to genetic drift within the population. “We’re talking about evolution in a Petri dish,” Dr. Jackson told GEN, and spontaneous mutations can give rise to subpopulations of genetic variants within the cell line. Immortalized and tumor cell lines may have an even greater proclivity toward genetic changes, because they often go through chromosomal rearrangements.

To monitor cell lines for genetic drift, David Yoder, Ph.D., a field application scientist at Thermo Fisher Scientific, recommends that scientists use STR assays that interrogate a broad range of STR markers including Thermo Fisher Scientific’s AuthentiFiler™, Indentifiler™ Plus, Indentifiler™ Direct, and GlobalFiler™. In addition to amplifying amelogenin, these PCR amplification kits contain primers for 9, 15, 15, and 21 different autosomal STR markers, respectively.

“Having a large panel doesn’t guarantee you will detect these [genetic] changes,” admitted Dr. Yoder, “but it does increase the opportunity to detect them.” Both GeneMapper® ID-X and GeneMapper® 5 Software, available through Thermo Fisher Scientific, allow users to process and compare STR data between samples to identify and track any significant changes in genotype throughout an experiment (Figure 4).

Figure 4. Thermo Fisher Scientific says that with the advent of higher throughput instrumentation and more robust amplification technology, laboratory bottlenecks have shifted from sample processing to data analysis. To expedite data review tasks, such as those related to genetic drift, the company has developed GeneMapper® software, which combines expert system and expert assistant capabilities.

Case of Mistaken Identity or Mismatch Repair

In addition to genetic drift and chromosomal rearrangements, many widely used cell lines, such as HEK293, CCRF-CEM, and Jurkat, have acquired mutations in mismatch repair genes. Commonly cited as a causative factor in cancer formation, these mutations compromise a cell’s ability to repair mistakes made during DNA replication. According to Markus Schmitt, Ph.D., founder and CEO of Multiplexion, cell lines with this genetic instability “have been shown to acquire changes in their STR profile upon long-term culture that can lead to false authentication results.”

Although profiling additional STR markers can improve authentication results for cell lines with deficiencies in their mismatch repair system, Multiplexion has developed an alternative approach for cell lines likely to be misclassified by STR profiling. Instead of using STR loci, the company’s Multiplex Cell Authentication (MCA) service uses a multiplex assay to simultaneously test 24 single nucleotide polymorphism (SNP) loci in the genome to authenticate human cell lines.

“Multiplexion’s cell line authentication system is a cost-effective alternative to STR profiling,” commented Dr. Schmitt. Currently, Multiplexion’s “steadily growing” database includes 850 of the most frequently used human cell lines.

Data from cell-based experiments forms the foundation for preclinical studies that lead to advances in disease prevention and treatment. After 50 years, cell line contamination and misidentification continue to undermine that foundation. Despite efforts to raise awareness and to make methods for cell line authentication accessible, the majority of scientists working with cell lines continue to forgo this important quality control test. Discussions around the barriers preventing scientists from adopting cell line authentication protocols are in progress, but it may take external pressure from grant-funding agencies and scientific journals to reinforce the integrity of scientific studies.

1. J.J. Boonstra et al., “Verification and Unmasking of Widely Used Human Esophageal Adenocarcinoma Cell Lines,” J. Natl. Cancer Inst. 102(4), (2010).
2. R. Harris, “Mistaken Identities Plague Lab Work with Human Cells (radio broadcast episode),
(December 9, 2014), accessed October 20, 2017.
3. L.P. Freedman et al., "The Economics of Reproducibility in Preclinical Research,” PLOS Biol. 13(6), (2015).
4. L.P. Freedman et al., “The culture of Cell Culture Practices and Authentication—Results from a 2015 Survey,” BioTechniques 59(4), (2015).

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