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Speciation of Animal Cell LinesSpeciation of Animal Cell Lines

Species Identification of Animal Cell Lines

Thanks to negative publicity initially prompted by a series of DSMZ publications highlighting the phenomenon, cell line cross contamination (CLCC) is now accepted as a major problem in cell culture. CLCC usually involves the seeding - presumably via shared pipettes, reagents etc - and eventual replacement of fledgling “new” cell line cultures by faster growing established cell lines. While short tandem repeat (STR)-DNA profiling detects cross-contamination among human cell lines, detection among animal cell lines at the DSMZ  is now performed by DNA-barcoding, a method involving PCR-amplification and sequencing of mitochondrial  DNA, followed by comparing the sequence in international data bases.

A fundamental requirement of cell lines is accurate characterization and exclusion of cross-contamination, whether by microorganisms or other cells. Cross-contaminated or misidentified cultures might yield invalid data and waste scarce resources (1).

First reports on cross-contaminated cultures appeared soon after the worldwide spreading and utilization of cell lines, as exemplified by the “HeLa-story” (2, 3). Although this problem may be chronic, and the scientific community adequately warned about this “hidden danger” (4-6), the need for authentication seems remains insufficiently appreciated rendering quality controls an essential part of current good laboratory practice (1, 7-9).



The Past

At the DSMZ, isoenzyme analysis, partially supplemented by cytogenetics, was used for identifying species in mammalian cell lines until 2000 (10). Since then, we are using DNA-bases methods. While DNA-fingerprinting techniques are used for the detection of individual human samples (11, 12) and as an international standard for authentication of human cell lines (13-15), animal cell lines derive from genetically homogeneous lab-bred individuals and require a different approach. One early study described PCR-assay using a single (alu) primer for distinguishing mouse and Chinese hamster cell lines (16). Based on this work, the DSMZ and other cell repositories have developed a PCR-based method for verifying species in commonly used cell lines (17-20). Our first approach was to introduce informative primer sets targeting exon or intron sequences of the β-globin and β-actin gene verifying the presumptive origin species of human, mouse, rat and hamster (17).

Also, we developed a PCR, with only one primer pair of repetitive sequences for hamster, enabling us to distinguish in one assay the above mentioned rodents plus human cells (21; Figure 1). By using fluorescence-labelled primers in connection with capillary electrophoresis, the length of the PCR-amplicons can be determined more precisely. Also, since the detection limit of capillary electrophoresis compared to standard agarose gel electrophoresis is much lower, even minute amounts of the respective DNAs can be detected (Figure 2).

Figure 1: Single PCR targeting hamster repetitive sequences differentiating the most frequently used cell lines
Figure 2: Electropherogram of a capillary electrophoresis after PCR with fluorescent labelled primer targeting the Syrian hamster
Figure 3: Relative percentages of animal cell lines at the DSMZ

In life sciences, the most ubiquitous species of origin are rodent and human (Figure 3). However, for cell lines used in other disciplines, for example in veterinary medicine or in biotechnology, it is equally important to verify domestic animal species and to exclude cross contamination with other (animal) cell lines.



Figure 4: Multiplex-PCR with bovine, horse, ovine and porcine primer pairs. Lanes 1,5: pig; lanes 2,6: bovine; lanes 3,7: ovine; lane 4: horse; lane 8: donkey; lane 9: chicken; lane 10: pig; lane 12: water.

Since the mitochondrial genomes of a large number of animals are sequenced, it was obvious to develop PCR assays targeting mitochondrial DNA to evaluate samples of a larger number from different species. Like colleagues from Japanese and US cell banks (22, 23), we applied standard and multiplex-PCR for the analyses of the number of almost twenty animal cell lines (24; Figure 4 as an example of some livestock animals).




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The Present: DNA Barcoding

The development of fast and robust sequencing of even tiny amounts of DNA initiated a new area of taxonomy combining PCR amplification and sequencing. By using degenerative primers a  ca. 650 base-pairs region (the ”barcode”) of the mitochondrial Cytochrome C Oxidase I (CO1) from numerous taxa, including “all” species of fish, amphibians, reptiles,  birds, mammals and even insects can be amplified and subsequently sequenced.  The data are then compared with those of a public sequence database to assign the unknown sample to a known species. By now, all deposited animal cell lines have been re-evaluated by the barcoding method and almost all of the originally supposed species have been confirmed. In case of the very low number of the unmatched species, the database suggested a (very) close related species or even a subspecies.

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