Detection and Prevention of Cell Line Cross-Contamination | Biowire Spring 2012

By: Edward Burnett, Ph.D.; James Cooper, Biowire Spring 2012, 19–21

European Collection of Cell Cultures (ECACC)
Health Protection Agency Culture Collections, Microbiology Service Division
HPA Porton Down, Salisbury, Wiltshire, United Kingdom

Biowire Spring 2012 — Live Cell Imaging of Signaling Pathways

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Since its inception in the early 20th century1, cell culture has become adopted in almost every field of life science research2. Soon after the establishment of the first human cancer cell line (HeLa) in the early 1950s3, it was realized that vigorous lines could contaminate and overgrow slower-growing cultures. The problem of misidentified cell lines, however, is still prevalent.

HeLa’s success as a cell line, coupled with its immortality and vigor, led to its worldwide distribution during a period when cell culturists were perhaps oblivious to the ease with which crosscontamination could occur, particularly in the absence of good cell culture practices.

HeLa contamination was recognized as early as 1967. Using Isoenzyme Analysis, Stanley Gartler reported that 18 cell lines of supposed independent origin shared a rare enzyme isoform with HeLa. These included the Chang Liver, Hep-2C, and KB cell lines. Yet in the last decade, there have been over 1,000 citations for these cell lines4. It is estimated that 15–20% of cell lines currently in use may not be what they are claimed to be5. Astonishing as it may sound, a report in 2004 suggested widespread lack of vigilance in cell sourcing and identity testing6. Out of 440 respondents, over one third received their cell lines from other laboratories and almost half did not perform identity testing. There is no excuse for this. A literature search will soon reveal the available resources, with culture collections being particularly proactive on this subject7–10. The European Collection of Cell Cultures (ECACC®) for example hosts a comprehensive list of misidentified cell lines on its website:

Karyotyping, Isoenzyme Analysis, and, more recently, DNA Barcoding11 are invaluable tools for the detection of inter-species crosscontamination. Short Tandem Repeat (STR) profiling has become the standard for the intra-species identity testing of human cell lines. The assay works on the same principle as the forensic DNA fingerprinting technique developed by Sir Alec Jeffreys12. However, labor-intensive Southern blotting has now been superseded by a multiplex PCRbased technique that simultaneously amplifies the polymorphic STR loci in the genome. Each allele occurs in the population with a particular frequency. By amplifying a sufficient number of different alleles and multiplying by the frequency in which each occurs within the population, a unique profile for a cell line is obtained. As recently as 200813, 40 human thyroid cancer cell lines were analyzed by STR profiling. Only 23 unique profiles were obtained and many of the cross-contaminating cell lines were not even thyroid in origin. These cell lines had been previously used for two decades in the field of thyroid cancer research.

At this moment in time there is no standard STR protocol for the unique identification of non-human cell lines. ECACC is unique among culture collections in having uniquely fingerprinted its nonhuman cell lines with Jeffreys probes. There are initiatives in place to replace this technique with up-to-date molecular methods.

Cell culture collections have started to forge close relationships to tackle the issues of misidentification and are working together to publish a standard (American National Standards Institute [ANSI] ASN-0002) for the authentication of human cell lines, using STR profiling and a free-to-use online interactive database containing the STR profiles of almost 2,700 human cell lines for users to compare their STR data. Providing the closest matches, the database allows the identification of a cell line or alternatively gives confidence in the uniqueness of a novel cell line:

In his 2007 article entitled “Eradication of cross-contaminated cell lines: A call for action,”14 Roland Nardone proposed that cell line authentication should be conditional for the receipt of grant funding and for the publication of research findings. Although authentication is not yet a requirement for all funding or publication, things are slowly moving in this direction with a number of journals adopting an authentication policy. Requests for ECACC’s STR profiling service have increased significantly over the last two years. Occasionally, driven by a requirement from a journal to provide evidence of cell line identity prior to publication, samples are submitted after project completion. Often in these cases, the news is not good, and sadly there are more than enough examples of inability to publish due to the unwitting use of the wrong cell line. It is in the opinion of these authors that neglecting to perform regular identity testing constitutes poor cell culture practice and that the promotion and insistence on identity testing should be an integral part of cell culture training.

While considering the major advances in identification methods and the need for regular testing, it is worth reflecting on the good practices2,15 that can minimize the chances of cell line misidentification. Cell lines should be sourced from reputable suppliers who can provide certificates of authenticity. The gifting of cell lines should be avoided and novel human cell lines must be authenticated to ensure that they are indeed unique.

A continuous cell line has the capacity to multiply indefinitely, and if the cell line is not subject to a controlled cell banking regime of Master and Working Cell Banks then the chances of contamination, cross-contamination, and genetic and phenotypic instability increase with each successive subculture.

There are two main routes to cell line misidentification. Firstly, the accidental inoculation of one cell line with another. A faster-growing line, introduced by a single drop of cell suspension or the accidental reuse of a pipette, will soon completely displace the original culture. To avoid such scenarios, a policy of clearance and segregation is recommended as follows:

  • Only one cell line should be handled in a bio-safety hood at any one time, and the workspace thoroughly cleaned between cell lines.
  • Dedication of equipment to single cell lines — bottles and aliquots of cell culture medium and other reagents — must be enforced.
  • Bottles and aliquots should be clearly labeled and never shared between cell lines.
  • Waste pots must be regularly emptied and replenished with a disinfectant effective against both microbes and cell cultures.

The second and possibly more common route of cross-contamination is the accidental mislabeling of a flask or other container. Repeated hand-labeling can rapidly lead to cell line name corruption with letters being confused with numbers, etc. Throughout its cell banking operation, ECACC uses pre-printed labels detailing the cell line name and other relevant information. All labels are removed from spent flasks, reconciled, and attached to the cell culture records.

The regular checking of cell line morphology and growth characteristics by phase contrast microscopy and comparison with reference images can give an early indication of problems.

Identification of unexpected morphology or growth characteristics in a cell line soon after resuscitation from frozen storage could be an indication of either mislabeling the vials at the time of cryopreservation, or even the removal of the wrong vial from storage. Printed labels including the cell line name in its correct format must have adhesive suitable for low temperature storage, as inappropriately adhered labels will detach, leaving an unlabeled vial in the inventory. Users then have to rely on the accuracy of the inventory records and confidence that the unlabeled vial contains the expected cells. A combination of regular identity testing and vigilance of good cell culture practices is required to provide valid cell cultures to application and to assure that research is acceptable for peer review. Ignorance of this guideline will lead to an undesirable legacy and invalid research.



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  11. Cooper JK, Sykes G, King S, et al. Species identification in cell culture: a two-pronged molecular approach. In Vitro Cell Dev Biol Anim. 2007;43(10):344–351.
  12. Jeffreys AJ, Wilson V and Thein SL. Individual-specific fingerprints of human DNA. Nature. 1985;316:76–79.
  13. Schweppe RE, Klopper JP, Korch C, et al. DNA profiling analysis of 40 human thyroid cancer cell lines reveals cross-contamination resulting in cell line redundancy and misidentification. J Clin Endocrinol Metab. 19 Aug 2008. 14 Feb 2012. (
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