Don Finley Sigma-Aldrich

Make sure your cell lines are authentic and uncontaminated by asking these simple questions.

In 2008, a pediatric research lab published in the journal Clinical Cancer Research exciting evidence of a therapeutic target to suppress invasion and metastasis in osteosarcoma. The survival rate of osteosarcoma patients had not improved for more than 20 years, which made the newly identified target particularly compelling because pharmacological inhibitors for it already existed.1

However, five years and nearly 80 citations later, the authors retracted the paper in 2013. DNA fingerprinting had revealed that the cell lines, OS187 and COL, were actually the widely used NCI60 colon cancer line HCT 15 and a human neuroblastoma cell line, respectively. Further investigation revealed that a vial of OS187 dating back to the lab’s second passage of the cells in 2002 also was misidentified.2

This retraction is only one of many similarly heartbreaking setbacks that can derail the success of talented investigators. The impact of cell-line contamination extends beyond the bench. Promising results generated from the use of misidentified esophageal cell lines led to at least three NIH grants, more than 100 scientific publications, 11 U.S. patents, and patient recruitment for clinical trials.3

Asking a few simple questions of the source of your cell lines, be it a commercial vendor or colleague down the hall, and following several basic best practices can help ensure that cell lines are authentic and free of contamination.


  1. How was the cell line authenticated?
  2. Is the cell line found in the ICLAC Cross-Contaminated or Misidentified Cell Line Database?
  3. Is the short tandem repeat (STR) profile known? Does the vendor supply a Certificate of Analysis (CofA) showing the STR profile of a cell?
  4. Has this batch been tested for mycoplasma?
  5. Are references and journal citations available for the cell lines?
  6. Who can answer specific questions about the cell line’s properties, behavior at later passage numbers, and identification?


  1. Has a reserve of cells been created for each cell line?
  2. Do cell culture reagents, e.g., media and sera, meet your requirements for cGMP certification or testing for endotoxins, mycoplasma, and viruses?
  3. Is each cell line used with its own bottle of media?
  4. Does each scientist maintain a personal stock of reagents?
  5. When was the last time cells were tested for mycoplasma and cell identity authenticated?


  1. Are antibiotics necessary? If so, which antibiotics are present in the media? If multiple antibiotics are used, is the cytotoxic concentration of that combination lower than when used independently?
  2. What is the confluency range required to maintain a viable culture?
  3. Have newly acquired cell lines been properly documented, including source and number of passages?
  4. After how many passages does your cell line exhibit different characteristics that may affect the integrity of your experiments? At what passage will your lab discard cells in culture and thaw a vial of stock?
  5. Do you test cell cultures for mycoplasma contamination weekly or monthly?
  6. Do you test the identity of the cell line at regular intervals or at key points in a project?

1  Zhang, P., Yang, Y., Zweidler-McKay, P. A. & Hughes, D. P. Critical role of notch signaling in osteosarcoma invasion and metastasis. Clin. Cancer Res. 14, 2962–2969, doi:10.1158/1078-0432.CCR-07-1992 (2008).
2  Retraction: Critical role of Notch signaling in osteosarcoma invasion and metastasis. Clin. Cancer Res. 19, 5256–5257 (2013).
3  Boonstra, J. J., et al., Verification and unmasking of widely used human esophageal adenocarcinoma cell lines. J. Natl. Cancer Inst. 102, 271–274, doi:10.1093/jnci/djp499 (2010).

Don Finley ([email protected]) is a market segment manager for cell culture reagents and ECACC cell lines at Sigma-Aldrich. To read more recommendations from Sigma-Aldrich to improve reproducibility of experiments, visit its translational research website at:

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