Why the Future of Biological Research Requires Cell Line Authentication

This spring, Psomagen was excited to announce the recent launch of our new Cell Line Authentication (CLA) service, using advanced STR profiling. CLA is quickly becoming an industry standard for funding and publication, making this an important step in any research project.

In this article, learn the basics of cell line authentication, then see why more organizations are turning to CLA to ensure their sequencing results are accurate, reproducible, and publishable. 

What Is Cell Line Authentication? 

Cell line authentication, also called cell fingerprinting or short tandem repeat (STR) profiling, verifies the genetic content and identity of cell lines. STR profiling is the gold standard for cell line authentication. This technology is used to compare samples, either to a reference genome or another sample, by matching up small sections of DNA occurring at specific locations in the genome. 

Why is cell line authentication important? 

Cell line misidentification or contamination can happen in any lab. This creates a waste of time, money, and effort for researchers. Awareness of cell line misidentification grew significantly in the 1990s. STR profiling was introduced as a solution in 2001. However, the number of misidentified cell lines reported annually has continued to rise (Figure 1). An estimated 18 to 36% of popular cell lines are misidentified. 

Fig. 1 : The distribution of the contaminated primary literature over the years.

In notable cases, cell misidentification has led to publications being retracted. For example, a 2010 Nature Methods paper by Ivan Radovanovic et al. was retracted after contamination with HEK cells expressing GFP was discovered in the majority of their gliomasphere lines. The case was a shocking reminder of how easily cell line errors can undermine years of research.

In response, many journals and funding sources have begun to require cell line authentication prior to article submission or grant application. Both the National Institutes of Health and the Food and Drug Administration have issued requirements and guidelines for cell line authentication in projects they fund. 

Which scientific publishers require cell line authentication? 

The International Journal of Cancer (IJC) has stated that approximately 4% of considered manuscripts are rejected from publication because of severe cell line issues. Many esteemed journals are beginning to implement CLA requirements to be considered for publication. 

Our lab maintains a list of CLA requirements. This list is ever-expanding as more scientific publishers recognize the need for cell line verification. At the time of this publication, major journals and publishing groups requiring CLA include:

• The American Association of Cancer Research (AACR) Publishers require cell line authentication on all cell lines used in submitted studies. 

• Nature Publishing Group strongly recommends cell line authentication, and encourages authors to submit certificates of authentication with their article submissions. 

• The Society for Endocrinology and the Endocrine Society now require cell line authentication for all lines used or described in a submitted study. 

• NIH publishers like the National Cancer Institute (NCI) require authentication of all cell lines and the date of last authentication. 

Psomagen’s Cell Line Authentication Process

Typical STR profiling follows a fairly standard set of steps. In Psomagen labs, the cell line authentication process looks like this: 

• Sample submission and gDNA extraction. Our labs accept fresh or frozen cells, dried cell pellets, fresh tissue, or pre-extracted gDNA. Typical CLA projects are looking at xenografts, cancer cells, or stem cells, but other options are possible. In the case of cell and tissue samples, a gDNA extraction step takes place prior to multiplexing.

• STR Multiplex PCR. Multiple target DNA regions are amplified during a single PCR reaction. For precise results, our labs use ThermoFisher's CLA GlobalFiler^TM kit, targeting 24 STR loci including 3 sex-determining markers.

• Sequencing and analysis. At Psomagen, CLA sequencing is conducted on the ABI 3730xl DNA Analyzer using GeneMapper software. Psomagen CLA services are performed in our CLIA-Certified, CAP-Accredited labs, meaning users 

• Data interpretation and reporting. Psomagen offers three service options: comparing against a single STR profile, against a database, or between two submitted samples. Based on this comparison, a matching percentage is calculated. Mismatched results may require additional analysis. 

• Report generation and delivery. All CLA reports come with a peak plot and allele table. We also provide guidance on what constitutes a strong match. Expert interpretation and analysis is also available. You can download a Tier 2 Sample Report here.

	Option 1: Single STR Profile	Option 2: Comparative with Database	Option 3: Comparative with Two Samples
Allele table (STR profile)	✓	✓	✓
Peak plot (Electropherogram)	included by request	✓	✓
Results saved for future comparison	✓	✓	✓
eSigned report		✓	✓
Matching % (comparison with public database or previously submitted sample)		✓	✓
Comprehensive interpretation of results (expert's comments)		✓	✓
Comparative analysis report between two submitted samples			✓

Why STR profiling?

Various techniques have been used to authenticate cell lines. These include cell morphology analysis, enzymology, cytogenetics (karyotyping, FISH), STR profiling, and SNP genotyping DNA sequencing. 

Among these methods, STR profiling is the most widely adopted and trusted approach, supported by the international standard ASN-0002 for human cell line authentication. This method is cost-effective and accurate, making it a popular choice for cell line validation. 

Why use 24-Plex STR for cell line authentication? 

ANSI/ATCC ASN-0002-2022 guidelines recommend 13 STR loci with 1 sex-determining marker for testing. However, an expanded test may be a better choice for your project. At Psomagen, our CLA test analyzes 24 loci. This includes the 13 loci recommended by ANSI/ATCCi and 11 additional markers, including 3 sex-determining markers (Table 1).

This expanded test offers superior discrimination for cell line authentication, lowering the Probability of Identity (POI). This makes it significantly less likely for different cell lines to share the same STR profile. 

Additionally, our CLA test uses a 6-dye system to significantly enhance the resolution of STR profiling and provide clearer differentiation. This leads to more accurate and reliable identification, even with challenging or degraded DNA samples, making it an ideal solution for precise cell line verification.

When Should You Authenticate Cell Lines? 

Cell line authentication can be conducted at several timepoints in a study. Experts recommend conducting CLA: 

• When submitting a grant application in alignment with NIH requirements. 

• Before submitting your manuscript to scientific journals. 

• When acquiring a new cell line. 

• When creating new cell lines. 

• Every 10 passages of cell culture. 

• As part of quality control to maintain cell integrity. 

• As a control measure when beginning a new study. 

• When observing inconsistent, unexpected, or irreproducible results. 

• Before freezing stocks or creating working cell banks. 

Cell line authentication is an important tool to ensure your biological studies are conducted with confidence and accuracy. As we uncover more about the risk of misidentification and cross-contamination, more and more funding sources and publishers will add CLA requirements to prospective studies. 

Psomagen’s CLA offers three tiers of analysis, expedited turnaround options, and detailed reporting. Our workflows are compatible with pharma, biotech, and academic workflows, and are fully compliant with funding organizations using NIH standards. You can learn more about this service in our labs, or visit our order site to get started today.