The Ongoing Importance of Cell Line Authentication

Cell lines are indispensable tools in both academic and industrial laboratories worldwide, forming the foundation of many breakthrough discoveries. However, cell line misidentification represents a longstanding and pervasive problem in basic and translational research that poses a significant threat to research reliability, integrity and reproducibility.

Studies estimate between 18-36% of cell lines utilized in biomedical research are contaminated and/or are completely misidentified1. As of January 2023, the ICLAC Register of Misidentified Cell Lines has documented 582 instances of cell line misidentification. Additionally, a study aimed at authenticating 278 human tumor cell lines used in China found that nearly 46% of the samples were either cross contaminated or misidentified2.A cell line becomes misidentified through cross-contamination, mislabeling, or other laboratory errors, meaning that it no longer corresponds to the original donor and may in fact come from an entirely different species, tissue or disease3.

Embarking on a project with a cell line without confirming its identity poses substantial risks for investigators. Studies carried out with misidentified cell lines is not only a waste of time and money, it can add misinformation to the literature and spur additional studies based on erroneous data that are also of questionable value. Documented cases of researchers having to retract their work due to contaminated or misidentified cell lines serve as cautionary tales1,3. And untold numbers of industry researchers have incorrectly prioritized programs based on misleading preclinical data generated from misidentified cell lines, which is disastrous for any biopharmaceutical company focused on filling its pipeline. As a result, many journals and funding agencies now require researchers to authenticate their cell lines prior to paper or grant submission using an accepted consensus method, such as Short Tandem Repeat (STR) profiling1,3,4,5.

For academic researchers, biopharmaceutical companies, and institutional core facilities alike, implementing rigorous quality control practices to ensure the identity of cell lines used in research can enhance the reliability of preclinical data to support the successful translation of research findings into clinical applications.

Short Tandem Repeat Profiling is the Gold Standard for Cell Line Authentication

In 2011, a committee of experts convened by ATCC (American Type Cell Culture Collection) introduced the ASN-0002 “Authentication of Human Cell Lines: Standardization of STR Profiling” consensus guidelines. This document, later revised in 2022 (ANSI/ATCC ASN-0002-2022), provides comprehensive guidance on the use of STR analysis for human cell line authentication.  ANSI recommends 13 autosomal STR loci as a standard for authentication. Today, STR profiling remains the gold standard methodology for cell line authentication (CLA)3,4,5.

STRs are short (2–7bp) repeating sequences of DNA scattered throughout the genome that provide a powerful tool for cell identification and human sample matching because the number of repeated units varies significantly between individuals. The STR profiling process uses polymerase chain reaction (PCR) amplification of multiple STR loci from the genomic DNA, followed by fragment analysis using capillary electrophoresis (CE), generating a unique genetic fingerprint for each cell line. This STR profile or fingerprint can be compared to a reference to verify the identity of a cell line. Organizations such as ATCC and Leibniz-Institute DSMZ (German Collection of Microorganisms and Cell Cultures) provide online access to searchable databases that allow investigators to query known cell types6. An STR profile with an 80% match or higher in profiled loci provides confidence that the cell line is authentic and has not genetically drifted from the original donor material, whereas in instances where the match falls between 55% and 80%, further investigation is recommended (ANSI/ATCC)3.

STR profiling as a quality control test offers several advantages: it is sensitive, highly reproducible, and relatively easy to execute. In fact, researchers can perform STR profiling independently using commercially available kits that include primers and reagents to allow co-amplification of multiple well-established STR markers in a single PCR reaction. This eliminates the need for primer design and PCR optimization, streamlining the authentication process.

Comprehensive Solutions for Cell Line Authentication: CE Instruments and Commercial STR Kits

ThermoFisher offers a variety of thermal cyclers such as the VeritiPro™ and ProFlex PCR system™ and genetic analyzers such as the Applied Biosystems SeqStudio™ and SeqStudio Flex Series that are optimized for CLA fragment analysis. Additionally, the Applied Biosystems™ product portfolio has several different kits for PCR-based STR fingerprinting for use on CE instruments and these kits cover the 13 loci recommended by ANSI (Figure 1)6.

  • The CLA Identifiler Plus PCR Amplification Kit has been optimized to analyze 16 highly variant human STRs over a wide range of purified gDNA preparations.
  • The CLA Identifiler Direct PCR Amplification Kit was developed to analyze the same 16 STR loci, starting from dried blood or buccal spots (for example, on NUCLEIC-CARD devices) or buccal swabs.
  • The CLA GlobalFiler PCR Amplification Kit can be used when extra levels of discrimination are needed, allowing for 6-dye analysis of 24 loci, 16 of which are included in the Identifiler kits.

The GeneMapper Software and the cloud-based microsatellite analysis (MSA) software solutions facilitate analysis of STRs by making use of pre-established allelic ladders and sizing bin sets for the various STR alleles covered by the Identifiler kits.

Figure 1. Workflows for cell line authentication. Two methods are available for cell line authentication. (A) Cells can be spotted onto NUCLEIC-CARD devices, 1.2-mm punches of the cards amplified directly without purification using the CLA Identifiler Direct kit, and fragments analyzed on Applied Biosystems CE instruments using GeneMapper Software 6 or MSA cloud application. (B) Alternatively, gDNA can be purified from cell lines, amplified using the CLA Identifiler Plus or CLA GlobalFiler kit, and fragments analyzed by capillary electrophoresis and GeneMapper Software 6 or MSA cloud application.

With these advanced instruments and dedicated reagent kits, Thermo Fisher has all the necessary tools for researchers to implement robust and reliable cell line authentication using STR profiling as part of routine quality control in their laboratories.

Using unauthenticated cell lines undermines the integrity of scientific research. By integrating cell line authentication (CLA) into standard laboratory practices, stakeholders involved in biomedical research can prove that the cell lines they have used are authentic and have remained so over the course of a study, providing confidence in accuracy, reliability, and translational applicability of the cell-based data generated. As stakeholders in the scientific community, we will continue to do our part to raise awareness and make methods for cell line authentication accessible to researchers worldwide, to address the persistent challenge of misidentified cell lines and reinforce the integrity of scientific research.

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