Human Cell Line Authentication Services

Bio-Synthesis, Inc. (BSI) offers fast and reliable human cell line authentication services, using STR DNA typing, to assist researchers in confirming species identity and detection of possible intra-species contamination or misidentification. This technology is/a provides:

Please contact us directly to discuss options that may be applicable to your project.

For decades, inadvertent cell line contamination has been a major issue for biomedical researchers. Cell line integrity is of utmost importance especially in stem cell, cancer tumor research and other new, developing research. In the culture of mammalian cells, laboratory personnel usually are more concerned about microbial (bacteria, yeast, mycoplasma) contamination, while intraspecific^{1, 2} and interspecific³ cross-contamination between different cell lines may be overlooked. The contamination and overgrowth of cell lines is a well documented, but often neglected phenomenon. Contamination by HeLa cells is only the best known example, and shows that any rapidly growing cell line can overtake both tumor cell cultures and normal cell lines. Nelson-Rees et al⁴ were among the first to document the extent of this problem. The implications are far reaching, including invalidation of published data and lost time, money and effort. As a result, NIH has recommended that all cell lines used in research undergo authentication to verify their identity (NIH Notice Number OT-OD-08-17) and stated that "(g)rant applications that fail to employ such practices would not be considered of the highest quality and such manuscripts would not fare well in the journal review process." See detailed lists of misidentified malignant hematopoietic cell lines.

About STR Technology

Most DNA present in the genomes of higher animals does not actually code for protein, as well as gene introns, regulatory and structurally important sequences, RNA genes, pseudogenes and sequences of questionable function are present. A significant part of non-coding DNA is comprised of repetitive sequences. The "mini- or microsatellites" are highly polymorphic zones inside these non-coding regions and characterized by reiterated DNA blocks, each containing individual sets of repeats⁵. The single-locus short tandem repeats (STR) are located at specific loci, while multi-locus STRs are spread throughout the entire genome^{6, 7}. Repeat-number-variability polymorphisms define the length of the individual specific "alleles", which can be analyzed by Southern blot or polymerase chain reaction (PCR) techniques. The analysis of STR at each single-locus site results in the detection of two DNA fragments differing in length unless the paternal and maternal alleles comigrate. In contrast, the analysis of the genome using a multi-locus probe, detects a large and complex pattern of DNA fragments (see figure). A similarly high level of information can be obtained by STR DNA typing when several highly polymorphic regions are analyzed simultaneously to facilitate a more reliable typing of individual cell lines⁴

Solution by Using STR DNA Typing

Molecular profiling by short tandem repeats (STR or microsatellite markers) is a fast, affordable and highly discriminating method to authenticate a cell line. Due to the highly polymorphic nature of each STR loci, every cell line will have a unique digital bar code that can distinguish it from other sources. A common set of STR loci are widely used for forensic applications and can be used for intra-species authentication.

The DNA typing process used at Bio-Synthesis involves simultaneously amplifying fifteen STR loci and the amelogenin gene in a multiplex PCR reaction (from Applied Biosystems AmpFℓSTR® Identifiler) capable of detecting as low as 500 picograms of genomic DNA (~ 15 diploid cells). The amplicons are separated by capillary electrophoresis and analyzed using Applied Biosystem Identifiler kit and GeneMapper software. The pattern of repeats results in a unique STR identity profile for each cell line analyzed. The profile can be used as a baseline for comparison with future tests. On discovering an untoward match between newly accessioned cell lines and an existing DNA profile, further independent approaches should be used to confirm cross-contamination, including independent DNA typing systems, such as VNTR or multi locus STR typing, as well as immunological and cytogenetic analyses.

See sample preparation and delivery

Cell line validation request form

References

1. MacLeod RAF, Dirks WG, Matsuo Y, Kaufmann M, Milch H, Drexler HG (1999). Widespread intraspecies cross-contamination of human tumor cell lines arising at source. Int. J. Cancer 83: 555-563.
2. Dirks W, MacLeod RAF, Jäger K, Milch H, Drexler HG (1999). First searchable database for DNA profiles of human cell lines: Sequential use of fingerprint techniques for authentication. Cell. Mol. Biol. 45: 841-853
3. Parodi B, Arescu O, Bini D, Lorenzini R, Schena F, Visconti P, Cesaro M, Ferrera D, Andreotti V, Ruzzon T (2002). Species identification and confirmation of human and animal cell lines: A PCR method. Biotechniques 32: 432-440
4. Nelson-Rees, WA, Daniels D, Flandermeyer RR (1981). Cross-contamination of cells in culture. Science 212: 446-452.
5. Jeffreys AJ, Wilson V, Thein SL: Hypervariable 'minisatellite' regions in human DNA. Nature 314: 67-73 (1985).
6. Zischler H, Nanda I, Schäfer R, Schmid M, Epplen JT: Digoxigenated oligonucleotide probes specific for simple repeats in DNA fingerprinting and hybridization in situ. Hum Genet 82: 227- 233 (1989).
7. Häne B, Tümmler M, Jäger K, Schleithoff L, Janssen JWG, Drexler HG: Differences in DNA fingerprints of continuous leukemia-lymphoma cell lines from different sources. Leukemia 6: 1129-1133 (1992).