A Biomedical Researcher's Guide for Analyzing Short Tandem Repeat (STR) Genotypes of Human Cell Lines and in Vitro Tissue Samples Using Three Standard Authentication Algorithms

Cell line authenticity is critical for producing reproducible and rigorously valid biomedical research. As of version 14 of its Register of Misidentified Cell Lines, released in 2026, the International Cell Line Authentication Committee (ICLAC) has identified 560 commonly used cell lines that are misidentified and for which there are no known authentic samples, and 48 samples of misidentified cell lines for which authentic samples have been found. The Cellosaurus Cell Line Knowledge Resource of August 2025 lists information about 122,819 human-derived cell lines, of which 1396 are "problematic cell lines." From extensive surveys of frequently used cell lines around the world, on average 22% of cell lines, or 2 of every 9, being used in laboratories are likely to be incorrect. Consequently, it is important that cell lines be authenticated (1) before starting a project utilizing them, (2) during their use in a project, and (3) after the project is completed to avoid publishing invalid and irreproducible information. The authentication of human cell lines is best achieved through analysis of short tandem repeat (STR) sequences in the genomic DNA. The resulting genotype data must be compared with those of reference genotypes in databases to determine whether they are derived from the original tissue sample or are misidentified. Cellosaurus release 54.0 contains 9063 STR genotypes for human-derived cell lines. This report with its Supporting Information, along with the accompanying workflow protocol article, are designed to provide an explanation of how to acquire and best apply three standard algorithms for correctly analyzing STR authentication genotyping data of human cell lines and cultured tissue samples. Unlike the revised Human Cell Line Authentication Standardization of STR Profiling (ASN-0002), this article is aimed at researchers, editors, and reviewers who need to understand how to interpret the resulting comparisons. This article is related to the accompanying protocol article entitled "Analytical Workflow Protocol for Determining the Authenticity of Human Cell Lines and Tissue Samples by Genotype STR Analysis" (Korch et al., submitted). © 2026 Wiley Periodicals LLC.