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Cell Line Identification and Authentication


The widespread use of cell lines in biomedical research is nearly universal, but a major issue has continued to plague the scientific community for over 50 years:  cell line contamination and misidentification.

To help address this challenge, NIST and the American Type Culture Collection (ATCC) have partnered to establish a Mouse Cell Line Authentication Consortium and are working with partners to test and validate NIST’s patented authentication method .

The consortium’s goals include the following:

  • Test the effectiveness of  19 mouse STR markers
  • Create a public STR profile database for mouse cell lines
  • Publish written consensus standards for mouse cell line authentication
Mouse cell line authentication
Figure 1. Left: Mouse. Right: Proof of mouse cell line



Mouse Cell Line Authentication – The scientific community has responded to the misidentification of human cell lines with validated methods to authenticate these cells; however, there are few assays available for nonhuman cell line identification. We have developed a multiplex polymerase chain reaction (PCR) assay that targets tetranucleotide repeats in the mouse genome consisting of primers that amplify nine mouse short tandem repeat (STR) markers.

Unique profiles were obtained from fifty inbred mice and six mouse cell lines which were used to determine the allele distribution for each STR marker. Correlations between allele fragment length and repeat number were confirmed with sequencing. STR genotypes for L929 and NIH3T3 cell lines were shown to be stable with increasing passage numbers as there were no significant differences in fragment length in samples of low passage when compared to high passage samples. In order to detect cell line contaminants, primers for two human STR markers were incorporated into the multiplex assay to facilitate detection of human and African green monkey DNA. Adoption of this assay would increase confidence in studies using mouse cell lines and could ultimately save time and reduce costs by identifying misidentified or contaminated cell lines in advance. In addition, NIST is working on multiplex STR assays for Chinese hamster ovary and rat cell lines.

graph showing peaks of STR profile
Figure 2: NIH3T3 STR Profile Electropherogram and Cell Image

Major Accomplishments

U.S. patent (No. 9,556,482 ) for an authentication method using NIST-identified short tandem repeat (STR) markers

Research studies and cell culture repositories have reported that 18-36 percent of all cell lines are contaminated, and the International Cell Line Authentication Committee (ICLAC) currently lists over  475 cross-contaminated or misidentified cell lines in its database. Error-ridden papers are frequently published and cited, and funding and resources can be wasted on research projects developed with inaccurate findings (Stacey 2000, Clement 2013, Jia 2014). Based on only two contaminated cell lines (HEp-2 and INT 407), it has been estimated that approximately $713M has been spent on work that produced 7,125 publications using these erroneous cell lines.

Researchers can verify their cell lines using the same technique applied in forensics for identifying people – by profiling DNA through short tandem repeats (STRs). Due to the success of STRs for human identification, this method has been applied to many human cell lines used in research (Masters 2001, Lorenzi 2009), and these results are now available in public access databases of authenticated DNA profiles. A lab can compare most of the human lines they use with this published data.

Methods for identifying non-human species have not been as straightforward since unique STR markers for distinguishing non-human cell lines have not been available. NIST has now identified a series of STR markers that can be used to identify  African green monkey and mouse cell lines, both of which are widely used models for studying human disease. We've also identified 11 STR markers specific to Chinese hamster ovary (CHO) cell lines, which are instrumental in the manufacturing of recombinant protein therapeutics, and successfully used eleven of those markers in a multiplex polymerase chain reaction (PCR) assay.

We are currently developing a PCR-based multiplex assay for use in authenticating rat cell lines. The development of these STR markers, and collaboration with companies that can develop commercial tests from them, will help to establish reliability of cell lines used in research. 

Human Cell Lines Standards and Protocols – As a prerequisite for manuscript submission and funding, many journals and funding agencies now recommend or require cell line authentication. In the National Institute of Health’s (NIH) Notice Number: NOT-OD-15-103 "Enhancing Reproducibility through Rigor and Transparency", released June 9, 2015, NIH revised its grant application instructions to include regular authentication of key biological and/or chemical resources. Specifically, the agency stated that, beginning in January 2016, “NIH expects that key biological and/or chemical resources will be regularly authenticated to ensure their identity and validity for use in the proposed studies.”

The current gold standard for human cell line authentication is Short Tandem Repeats (STR) genotyping. ANSI/ATCC consensus standard ASN-0002 “Authentication of Human Cell Lines: Standardization of STR Profiling” details all aspects of human cell line authentication using STR analysis. Commercial kits are available for genotyping; various companies offer genotyping services for human cell line authentication, as well.

National repositories list at least eight (8) human STR markers and amelogenin (AMEL), the sex determination marker for each human cell line deposited. Human STR profile databases (ATCC, DSMZ, CLIMA, NCBI) are available for human cell line data comparison. 


Created July 7, 2015, Updated October 23, 2018