Cell Culture Contamination

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Cell Culture ContaminationTable of Contents



Don Finley
Don Finley

Life science research using cell cultures has resulted in numerous improvements to the treatment of human disease. In addition, with the advent of stem cell technology, cultured cells are themselves becoming therapeutics. In this light, cell culture contamination could be one of the biggest hindrances to scientific progress today.

Researchers typically think of cell culture contamination in terms of microorganisms such as bacteria, fungi, viruses, and protozoa, but contamination can also be caused by chemical contamination as well as contamination by other cell lines — for example HeLa cells. The difficulty in controlling a particular contaminant is often related to its ease of detection. Most bacterial and fungal contaminations will cause the medium to turn cloudy and quickly kill the cells in culture. Although losing cells and any data relating to the current experiment is never desirable, the damage, however severe, is limited. Comparatively, viruses are difficult to detect. They cannot be detected by conventional light microscopy and may not be detectable by electron microscopy if they are integrated in the host cell genome. However, viruses have stringent requirements for the type of cells they can infect and, as they often result in the death of the cell lines they infect, are self limiting and are much rarer than other types of contamination. The two most worrisome contaminants researchers face today are mycoplasma and cross-contamination and/or misidentification of cell lines.

Mycoplasma are very simple bacteria that do not possess cell walls. As such, they are not killed by typical antibiotics used in cell culture, and are not detectable by conventional light microscopy. In addition, they can grow to very high concentrations in cell culture without causing the media to become cloudy as is seen with other microorganisms. Since mycoplasma have evolved as parasites they are difficult to culture in the absence of mammalian cells. They do not usually kill the mammalian cells they contaminate, but can greatly influence the cells they infect by altering cellular metabolism, causing chromosomal aberrations, slowing cell growth, and interfering with cell attachment. n short, they are likely to influence the results of most experiments performed using affected cell lines. Contaminations can be undetected for long periods of time, and if left unchecked they can contaminate an entire operation or facility.

Misidentified or cross-contaminated cell lines in some ways are the most concerning problem of all. Research based on the premise that, for example a cell line is representative of human colon cancer when in actuality it is an ovarian cancer cell line or perhaps a cell line from a different species, can call the results of that research into question. Considering that a researcher may spend many years performing research using a particular cell line and publish numerous papers on that research, it is easy to understand why this problem has many scientists very uneasy.

How big is the problem of contamination? Based on studies by FDA, ATCC, and others, it is estimated that 11% to 15% (or more) of all cell cultures today are contaminated with mycoplasma. Furthermore, over the past 25 years, numerous studies combined with the experiences of various cell culture repositories, indicate that 18 to 36% of cell lines in existence today are misidentified1. This level of contamination and misidentification seems ironic, given that the causes of cell culture contamination are well understood; the methods of prevention, detection, and elimination of mycoplasma are commonplace; and a variety of methods for cell line identification, such as STR analysis, isoenzyme studies, and DNA fingerprinting are readily available.

It is thought that some labs are reluctant to test for cell line contamination for fear that the results could call into question many years of work, and lead to publications being retracted because of erroneous data. As understandable as this reluctance might be, ultimately we all are affected. Erroneous, misinterpreted data not only are economically wasteful but ultimately slow scientific progress. There are a growing number of scientific publications calling for scientists to get control of cell culture contamination and ensure cell lines are correctly identified prior to publication2-9. Unfortunately, this is not yet a requirement for publication, and scientists continue to publish using cell lines without first verifying their identity or testing for mycoplasma or other contaminants.

It has been suggested that the high levels of contaminated cultures seen relate primarily to labs lacking a good plan to minimize and test for contamination and cell line mix-ups, as well as a failure to adhere to good aseptic cell culture techniques. Labs that have in place a good plan to reduce cell culture contaminations will usually have a much lower incidence of cell contamination than those labs that do not have such a plan.

The key thing to remember is that while contamination cannot be completely eliminated it can be minimized and controlled. At Sigma, we have in place stringent process controls and we extensively test to ensure the products we offer for cell culture are contamination free. Our line of ZFN-engineered cell lines and the ECACC cell lines we offer are well characterized, correctly identified, and are free of contaminants. We test our cell culture sera, media, and reagents for endotoxin and bacterial contamination, and offer many animal component-free products to eliminate potential risks of TSE and animal virus contamination.

The products included in this Biofiles issue represent some very useful tools to help make contamination control easier for researchers. We offer a comprehensive line of antibiotics that, when used appropriately, reduce the chances of unwanted contamination. We have a number of products to help reliably, effectively, and economically detect and if necessary eliminate mycoplasma contamination from cultures. The Labware products in this issue are useful in contamination control and detection, and include microscopes, glove bags, and sterile plasticware.

Ultimately, we at Sigma feel strongly that easing the risk of cell culture contamination is part of our mission in research cell culture. In addition to products, we provide a wealth of technical information, both on-line and in print, to help you reduce your risk of contamination. To view our on-line version and to request a copy of our Sigma-ECACC Fundamental Techniques in Cell Culture Laboratory Handbook, please go to: sigma.com/ecacchandbook.

To request a copy of our 2011–2012 Cell Culture Manual, please go to sigma.com/cellculturemanual.


  1. Editorial, Identity Crisis, Nature 457, 935-936 (2009).
  2. Podolak, E. Ending Cell Line Contamination by Cutting off Researchers, Biotechniques Online News (2010).
  3. American Type Culture Collection Standards Development Organization Workgroup ASN-0002. Cell Line Misidentification: The Beginning of the End. Nature Reviews 10:441-448 (2010).
  4. Nardone, R.M. Eradication of Cross-contaminated Cell Lines: A Call for Action. Cell Biology and Toxicology 23:367-372 (2007).
  5. Nardone, R.M. Curbing Rampant Cross-contamination and Misidentification of Cell Lines. BioTechniques 45:221-227 (2008).
  6. Harald zur Hausen, Obligation for Cell Line Authentication: Appeal for Concerted Action. Int. J. Cancer: 126 1 (2010).
  7. Chatterjee, R., Newsfocus, Cases of Mistaken Identity. SCIENCE VOL 315 16 (2007).
  8. Capes-Davis, A., et al., Check Your Cultures! A List of Cross-contaminated or Misidentified Cell Lines. International Journal of Cancer. 127:1-8 (2010).
  9. Phuchareon, J. et al., Genetic Profiling Reveals Cross-contamination and Misidentification of Six Adenoid Cystic Carcinoma Cell Lines: ACC2, ACC3, ACCM, ACCNS, ACCS, and CAC2. PLoS ONE 4:6 (2009).

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