Verifying Cell Lines through Authentication

Experimental success corresponds directly to the quality and conditions of the cell lines used. Cells that are kept too long in culture and are not periodically tested for genotypic or phenotypic stability may no longer be reliable models of the original source material.

To maintain high cell culture standards and ensure reliable, reproducible results, the use of authenticated and quality-tested cell lines from a recognized cell bank is highly recommended.

ATCC authenticates cell lines routinely with the following tests:

STR profiling establishes a DNA fingerprint for human cell lines

ATCC STR profiling uses multiplex PCR to simultaneously amplify the amelogenin gene and eight of the most informative polymorphic markers in the human genome. The pattern of repeats results in a unique STR identity profile for each cell line analyzed. STR analysis is critical for verifying the identity of human cell lines and is performed for each distribution lot. The results are compared to the baseline profile of the token stock derived from the depositor.

STR STR
STR STR

STR profile of two unrelated cell lines. Top: KU812E (ATCC® CRL-2100™). Bottom: MRC-5 (ATCC® CCL-171™). Amplicons are generated using Promega PowerPlex® 1.2 system, separated by electrophoresis and analyzed using Taqman® Genotyper 2.0 software from Applied Biosystems.

Cell morphology is monitored throughout all processes

Cellular morphology can vary between lines depending on the health of the cells and, in some cases, the differentiation state — a critical property in certain assays. Morphology can change with plating density as well as with different media and sera combinations. Morphologies of cells grown at low and high densities at ATCC are recorded and used routinely to check cell lines during accessioning and bioproduction.

CCL-1 High Densirty

ATCC® CCL-1™ at high cell density

CCL-1 Low Density

ATCC® CCL-1™ at low cell density

Karyotyping is performed to identify the species as well as variation within the cell line

Karyotyping is a basic and indispensable test performed routinely to determine if the line has maintained a stable genotype. Karyotyping is performed on all hTERT immortalized cell lines and on many ATCC classic cell lines.

CRL-4001_DI karyo CRL-4001_SEED karyo

ATCC® CRL-4001™ Giemsa-banding on distribution (left) and seed (right) stocks

The ATCC COI Assay is used to reliably determine the species of a cell line

The use of cytochrome C oxidase I (COI) testing at ATCC replaces isoenzymology in determining the true species of a cell line. The cytochrome C oxidase I gene (COI) is conserved genetic material found in the mitochondria among closely related species and across diverse phyla in the animal kingdom. 2* Based on the species-to-species sequence variability of the COI gene, ATCC scientists developed a PCR-based speciation assay by designing unique primer pairs that recognize only a specific species and produce amplicons in a multiplex PCR reaction with sizes no less than 20 base pairs apart.1 The ATCC COI assay is capable of distinguishing cell lines of pig, human, cat, Chinese hamster, Rhesus monkey, sheep, horse, African green monkey, rat, dog, mouse, rabbit, goat and cow origin. When the species of a cell line remains in question a ~650bp ‘barcode’ region of the COI gene is sequenced for verification purposes.

* For more information on the Barcode of Life initiative, please see: www.boldsystems.com

Figure 1B All species

Amplified fragments were detected by ethidium bromide staining on a 4% agarose gel. Lane 1 shows the 100 bp ladder. Lane 2 shows the multiplex performance of oligonucleotide pairs specific for the following 14 species: pig, human, cat, Chinese hamster, Rhesus monkey, sheep, horse, African green monkey, rat, dog, mouse, rabbit, goat, and bovine. The template for the reactions consisted of 0.5 -1.0 ng mixed DNA contributed from all of the species with primers in the master mix.

Selected References:

  1. Cooper et al. Species identification in cell culture: a two-pronged molecular approach. In Vitro Cell Dev Biol Anim. Nov-Dec; 43(10):344-51. Epub Oct 13. (2007)
  2. Hebert et. al. Barcoding animal life: cytochrome c oxidase subunit 1 divergences among closely related species. Proceedings of the Royal Society of London, Series B. (2003b)

PowerPlex® is a trademark of the Promega Corporation. TaqMan® is a trademark of Roche Molecular Systems.