The Importance of Cell-Line Authentication

Cell-line authentication is the process of verifying the identity of the cells used in your experiments. It often includes confirming that cell lines are derived from the correct species and donor, and that they are contamination-free. In this article, we provide a recap on why cell-line authentication is important and review some of the methods available.

Using unauthenticated cell lines can create major problems

The latest version of the ICLAC Register of Misidentified Cell Lines released earlier this year, lists a staggering 576 cell lines as being misidentified. Of these, 531 have no known authentic stock, 73 do not correspond to the original donor, and 67 come from a different species, while HeLa is the most common contaminant among 144 different contaminants that have been identified. “At a minimum, scientists using misidentified or contaminated cell lines will waste valuable time and resources on experiments until the error is uncovered,” comments Leta Steffen, Senior Applications Scientist at Promega. “If the error goes undetected, the resulting data can lead to incorrect theories, derailing scientific progress and risking both reputations and future funding.” ICLAC has documented several case studies in cell-line misidentification, including an analysis of publications that refer to Chang liver cells in the title or abstract; these cells are now known to be HeLa derivatives that are unsuitable as a model for normal liver, causing major repercussions within the research community.

Phenotypic drift is a red flag

“Phenotypic drift is often a good indicator that cells have some type of issue,” reports Fang Tian, Ph.D., Principal Scientist at ATCC. “It can result from inconsistent cell subculture, or stresses associated with too high or too low cell seeding densities, and is especially common in cultures composed of subclonal cellular populations due to tumor heterogeneity. However, phenotypic drift is far more likely to result from contamination, which may be introduced accidently through routine cell culture or even during cell-line establishment.”

Sheng Guo, Ph.D., Executive Director of Systems Biology at Crown Bioscience, adds that phenotypic drift can sometimes be traced to genetic drift, especially where a drug treatment is used to selectively enrich cells with certain genetic mutations or other features. “Based on our experience, phenotypic drift most frequently results from cell-line mislabeling or contamination,” he says.

STR profiling is the current gold standard for cell-line authentication

In 2011, a committee of experts published the ANSI/ATCC ASN-0002-2011 consensus guidelines for best practices in cell-line authentication based on short tandem repeat (STR) profiling; a revision (ANSI/ATCC ASN-0002-2021) was published earlier this year. STRs are short (2–7bp) repeating sequences of DNA that provide a genetic fingerprint of an individual; for cell-line authentication, they allow researchers to verify whether a human cell line matches the original donor or if cross-contamination with another human cell line has occurred.

“A main advantage of STR profiling is that it is highly reproducible,” explains Tian. “Results are comparable across different laboratories and from various passages of a cell line tested at different times. STR profiling also has extremely high discriminating power and, because it is an established technology, results can easily be compared against a vast database for quick and accurate verification of cell-line identity.” These features of STR profiling have led it to become the gold standard approach for cell-line authentication, although a drawback of the technology is its limited ability for authenticating non-human cell lines. In efforts to address this limitation, a recent collaboration between ATCC and the National Institute of Standards and Technology (NIST) focused on developing mouse STR profiling technology has identified 19 murine STR markers to date.

STR profiling can be out-sourced or run in-house

“Most commercial STR chemistries can be performed as multiplexes and use several dye channels for electrophoresis,” notes Steffen. “This allows for analysis of many STR loci at the same time, providing sensitive discrimination between cell lines from different donors.” Promega’s GenePrint^® 10 and GenePrint^® 24 Systems enable analysis of 10 and 24 STR loci, respectively, and are widely used by core facilities for cell-line authentication on a fee-for-service basis. Additionally, Promega has developed the Spectrum Compact CE System for researchers interested in performing cell-line authentication in-house. “The Spectrum Compact CE System gives researchers access to capillary electrophoresis for both fragment analysis—including cell-line authentication—and Sanger sequencing,” says Steffen. “In less than a day, it is possible to purify DNA from cultured cells, perform PCR using a multiplex STR chemistry like GenePrint^® 24, electrophorese the PCR products on the Spectrum Compact CE System, then compare the resulting profile to a high-quality database—such as the ATCC STR database—or the profile of the source cells.”

NGS-based SNP profiling

Single nucleotide polymorphisms (SNPs) are naturally occurring variants that, like STRs, can be exploited for cell-line authentication. Critically, by providing insights into variation at the single DNA nucleotide level, SNPs enable detection of intraspecies contaminants and verification of species-specific tumor models—neither of which STR profiling is able to do at sufficiently high resolution.

“Historically, SNP profiling has been restricted to processing only low sample numbers due to the cumbersome workflow and the complexity of monitoring multiple SNPs in parallel,” reports Guo. “However, with the advent of next-generation sequencing (NGS), it is now possible to achieve much higher accuracy, sensitivity, and throughput for a more detailed profile of the cell line under investigation.” Of the various NGS technologies now available, Crown Bioscience has chosen to use barcode deep NGS for cell-line authentication (a method that employs multiplex PCR to amplify hundreds of targeted DNA regions for sequencing at high-depth for several hundred samples simultaneously). “With this approach, we are able to identify both human and mouse samples with 100% accuracy,” says Guo. “We can also identify contaminants and determine the contamination ratio, as well as being able to estimate mix ratios for three or more cell lines. With organoids, homograft models, and xenograft models frequently being used for scientific research, authenticating these heterogeneous systems is becoming more important than ever.”

Table. Comparison of STR profiling and NGS-based SNP profiling. Table provided by Crown Bioscience.

Best practices are essential for reliable results

For cell-based data to be trusted, it is recommended that researchers follow best practices for cell-line authentication as established by ICLAC and defined in the ANSI/ATCC ASN-0002-2011 and ANSI/ATCC ASN-0002-2021 consensus guidelines. Steffen stresses that cell lines should always be obtained from high-quality sources and suggests checking cell line names against the ICLAC Register of Misidentified Cell Lines and common cell databases such as ICLAC.org, Cellosaurus, or COSMIC prior to use.

“Cell-line authentication should be performed whenever a cell line is obtained or established, whenever a large banking is carried out, and routinely throughout every study,” adds Tian. “Moreover, cell-line authentication is vital whenever a novel phenotype is observed.” Lastly, Guo highlights the value of maintaining a complete record of each cell line during its entire life cycle, including storage, passaging, and distribution of cells. “Ensuring you have the right cell line and that it is free from contamination avoids unreliable research results that can lead to erroneous conclusions; wasted time, effort, and money; and damaged reputations,” he cautions.

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