Authentication of Cell Lines

The frequency of cross-contamination (CC) affecting new cell lines was previously shown to be about 17%, mostly intraspecific, human-on-human CC. A key question for research, development, or production programs involving cell lines is whether a given cell line is authentic, the authentication method of choice being short tandem repeat (STR) DNA typing. The DSMZ has developed a comprehensive database of DNA profiles of STR loci for all human cell lines in the collection.

Microsatellites in the human genome harboring STR markers allow the identification of individual cell lines at the DNA level (1). Polymerase chain reaction (PCR) amplification of eight highly polymorphic microsatellite STR loci and gender are standard tools for screening the uniqueness of DNA profiles in an STR database (2, 3). In cooperation with other Biological Resource Centers, the DSMZ has piloted the most comprehensive DNA reference database of human cell lines hitherto (Figure 1). The database is linked to a simple search engine for interrogating STR cell line profiles. The online-verification cell line identity tool is available on our website at

STR DNA typing is used in accession procedures of new human cell lines and for verifying cell line identities after replenishment. DNA reference database scans are included within the customer service of DSMZ for authentication orders (

Cell Line Cross-Contamination – The Neglected Danger

The application of species markers including cell surface antigens and chromosomes first alerted users to the problem of interspecies misidentification in cell culture (4, 5). Subsequently, it was shown that intraspecies contamination of human cell cultures was also a danger, but which could be monitored by isoenzyme analysis in a few cases (6). After extending this approach to multiple polymorphic isoenzymes, the persistence of specific marker chromosomes in long-term passaged cell lines demonstrated the unique power of cytogenetics (7). Based on the detection of chromosomal markers, it was convincingly demonstrated that multiple cell lines were actually derived from one source, namely the HeLa cell line (8). Furthermore, cross-contamination among established cell lines was measured at 16–35% in the late 1970s (9).

Over the last decade, we have shown that 14-18% of human leukemia-lymphoma cell lines are false, having been cross-contaminated by their originators with older established cell lines (10-12; Figure 2). Our data showing widespread cross-contamination served to refocus attention onto the need to verify cell line authenticity, leading ultimately to joint efforts by major cell banks to agree on common standards permitting cell line data comparisons between cell repositories and, interactively, with users.

STR DNA Typing

STR typing of tetrameric repeats is now the gold standard for authentication of human cell lines (13). The ATCC, DSMZ, JCRB, and RIKEN cell line banks have each built their own STR databases based on the following specific set of STR loci: D5S818, D13S317, D7S820, D16S539, vWA, TH01, TPOX, CSF1PO and amelogenin (AMEL) for gender determination. AMEL has meanwhile become the most suitable gene for gender determination of samples of human origin (13). Using specific PCR primers, the sequence of the X-chromosomal version (AMELX, Xp22.1–Xp22.3) yields a 106 bp amplicon, while the Y-chromosomal gene (AMELY, Yp 11.2) yields a 112 bp DNA fragment, which may be easily separated by different electrophoresis techniques. Hence, samples from male sources will show two bands in a PAGE analysis (209 and 215 bp), while female-derived cell lines will show only one band (209 bp). The advent of fluorescent labeling of PCR primers permits the multiplexing of STR loci which may have alleles that fall in the same size range by labeling the overlapping loci with different fluorescent dyes that can then be resolved spectrally (Figure 3). The application of commercial multiplex STR kits should be carried out strictly following the specific manuals of manufacturers. Finally, the combination of eight STRs raises the exclusion rate sufficiently to allow the discrimination of one human cell line from another at the level of ~108.

Figure 3: Interspecies cross-contamination. An electropherogram of a nonaplex STR reaction obtained from genomic DNA of the cell line HELA (left) and a tetraplex PCR reaction for detection of rodent mitochondrial DNA (mtDNA) are shown. Dye signals in green show STR loci within human genes, while black peaks show STRs outside coding regions. Blue peaks show mtDNA from rodent cell lines of mouse, rat, Syrian or Chinese hamster. Numbers written above the STR loci indicate the respective alleles.

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