Addressing Cell-Line Contamination to Improve Data Reproducibility

Best Practices for Validing, Storing, and Handling Cell Culture Uphold Research Integrity

The contamination of cell lines by other cell lines or by microbes is a plague on the biomedical research community.1 Cell-line misidentification was first acknowledged nearly half a century ago upon the discovery of HeLa contamination of 18 cell lines.2 The problem persists to this day, as an estimated 15–20% of all cell lines used in biomedical research continue to be misidentified.3 A PubMed query of five cell lines identified in the 1960s as HeLa-contaminated (KB, HEp-2, Chang liver, Int-407, and WISH) found nearly 400 citations between March 2009 and February 2014. Despite peer review, many of the published articles still described these cell lines as “normal” human cells, not cervical cancer cells.

The reproducibility of research findings is greatly impacted by contaminated cells, but the influence of contamination extends far beyond the laboratory bench. For example, results generated from the use of misidentified esophageal cell lines led to at least three NIH grants, 100+ scientific publications, 11 U.S. patents, and patient recruitment for clinical trials.4 Moreover, these events could also damage patients’ and the public’s trust in scientific research.

Microbial contamination of cell lines poses a serious challenge to obtaining reliable research data. The most dangerous of microbial contaminants are mycoplasma. These microbes cause no discernible change in turbidity or pH even at high concentrations. Mycoplasma can induce abnormal behavior in cultured cells, including altered growth rates, morphological changes, chromosomal aberrations, and altered cell metabolism. Contamination is difficult to control because mycoplasma lack cell walls and thus cannot be treated with most antibiotics.5 If left unchecked, mycoplasma can contaminate an entire operation or facility.

Cell-Line Integrity

Asking a few simple questions before purchasing cell lines from a vendor can help ensure that the cells are authentic and free of contamination (Figure 1). A reputable vendor will be able to adequately address each of the following questions:

  1. Is the cell line authenticated?
  2. Is the cell line found in the misidentified cell-line database (that is, in the Database of Cross-Contaminated or Misidentified Cell Lines, which is compiled by the International Cell Line Authentication Committee [ICLAC])?
  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 every batch been tested for mycoplasma?
  5. Does the vendor supply references and journal citations for the cell lines?
  6. Is technical support available? Will specialists answer specific questions about cell-line properties and identification?

The vendor should also demonstrate that upfront measures have been performed to validate cell-line quality and health. Established cell repositories such as the European Collection of Authenticated Cell Cultures (ECACC) and the American Type Culture Collection (ATCC) have already performed extensive tests on the cell lines in their libraries; thus, purchasing cells from these organizations is secure.

Figure 1. The steps that should be taken to ensure cell-line authentication vary depending on how cell lines are sourced. Ideally, cell lines should be obtained from established repositories, such as those maintained by The European Collection of Authenticated Cell Cultures (ECACC). ICLAC = International Cell Line Authentication Committee; STR = short tandem repeat.

Validation Techniques

The consistent application of validation methods and proper laboratory techniques are necessary for maintaining reliable cell cultures upon receipt from a vendor. Establishing a laboratory-wide plan of action for when contamination is detected will minimize research disruptions.

Mycoplasma testing should be performed weekly to monthly, and cell-line authentication should be performed before the first use, every six months to one year, and when a mix-up is suspected. If cell lines are obtained from a colleague (this is not recommended), they must be tested for authenticity and the presence of mycoplasma before use. The more reliable route is to purchase cell lines from a reputable repository, such as ECACC or ATCC.

Mycoplasma Detection

Low levels of mycoplasma contamination are difficult to detect and may require two or more methods for detection.

Mycoplasma Culture: Mycoplasma culture is a standard method of detection. The FDA/European Pharmacopeia-approved protocol, the most sensitive method, relies on a selective and highly enriched growth medium on standard agar plates. Resulting colonies have a distinctive “fried egg” appearance, and a positive result using this method is conclusive proof of mycoplasma contamination. However, because this method does not detect all species, such as Mycoplasma hyorhinis, DNA staining and/or polymerase chain reaction (PCR) analysis are required to assure the absence of contamination.

DNA Staining: DNA staining relies on the Hoechst 33258 stain, which causes DNA-rich nuclei and any mycoplasma in the cytoplasm to fluoresce. Mycoplasma and mitochondria are easily differentiated because mycoplasma has 10 times the DNA content, and thus their fluorescence is much brighter. Commercial kits for DNA staining are available.

Using this method, false positives can be caused by cell detritus or cells undergoing apoptosis. False negatives are also possible, as this is the least sensitive method. Unlike the culture method, however, DNA staining will detect all types of mycoplasma.

PCR: PCR-based mycoplasma detection can be very sensitive, capable of detecting as few as 20 copies of a mycoplasma genome within a 2-μL sample. Mycoplasma detection is achieved by a primer/probe system that amplifies the highly conserved 16S rRNA operon coding region of the mycoplasma genome. PCR detection is effective for 19 species of mycoplasma, including M. hyorhinis, which is not detectable by the culture method. If a lab routinely performs PCR, a commercial kit will work well. If not, several services are available to perform PCR-based mycoplasma testing, for a fee.

Cell-Line Authentication

In 2012, ECACC cofounded ICLAC. ICLAC members include representatives from the ECACC and other international cell culture organizations, as well as respected scientific researchers committed to championing the importance of cell-line verification. The ICLAC maintains a searchable database of cross-contaminated/misidentified cell lines. The comprehensive ICLAC database is updated periodically and serves as a valuable resource to the research community, but retractions do occur. After searching the database, it is also worth querying Google for the cell line name and the keyword “retraction.”

Short Tandem Repeat Profiling (DNA Fingerprinting)

A short tandem repeats (STRs) is a repeating unit of two to six nucleotides  in a DNA sequence. The number of STRs at any locus is highly variable within the human population, and these variations are heritable. The ECACC, the ATCC, and other cell repositories use the ASN-0002-2011 Standard Method based on the Combined DNA Index System (CODIS), which analyzes STRs at 13 specific loci and several additional loci to determine human cell-line authenticity.

Commercial kits for STR profiling are available, but conclusive identification can be ensured by using a repository such as the one maintained by the ECACC or a core facility that specializes in this type of analysis. No standardized method yet exists to validate the identity of nonhuman cell lines; however, ATCC is currently working with the National Institute of Standards and Technology (NIST) on an authentication method using NIST-identified STR markers.

DNA Barcoding

DNA barcoding involves PCR amplification and DNA sequencing of specific regions of mitochondrial DNA. The cytochrome c oxidase subunit 1 mitochondrial region (COI) is the standard target for humans; the rbcL region is the standard target for plants. In these regions, the DNA sequence is the “barcode” that differentiates species, although some variation exists between individuals of the same species.

Storage and Handling

The proper storage and handling of cells and cell culture reagents, as well as good aseptic cell culture technique, can minimize contamination and thus improve the reproducibility of experimental data.

Storage Best Practices

  • Create a reserve of cells at earlier passages to serve as a cell bank.
  • Purchase media, sera, and other culture reagents that are endotoxin-free and manufactured under cGMP. Sera should be tested for mycoplasma and viruses.
  • Each researcher should maintain a personal stock of reagents, if possible, to reduce bacterial and cross-contamination.
  • Maintain a separate bottle of media for each cell line.
  • Purchase fresh, validated cells only. Do not accept cells from other laboratories. The most common source of mycoplasma infection in cell culture research is a previously infected culture.
  • Maintain a log of mycoplasma and validation testing. Retain CofAs for reference.
  • Cell lines should be stored below the glass point of water (at least −150 °C). Monitor nitrogen storage temperature and maintain a temperature log. Program an alert to sound if the temperature exceeds a certain deviation from the set temperature.

Handling Best Practices

  • Avoid distractions to prevent cross-contamination when working with cells.
  • Use antibiotic-free media unless undertaking primary culture. Overuse of antibiotics can lead to resistant bacterial strains.
  • Take care when using two or more antibiotics in the same culture, as the cytotoxic concentrations for the combined treatments are lower than those listed for the individual antibiotics.
  • Discard waste and spray hood with 70% ethanol after use. When multiple cell lines are in use, work with one cell line at a time and clean the hood before moving to the next cell line.
  • Do not allow cells to become fully confluent. Passage cells at 70–80% confluency or as advised by an ECACC or a ATCC data sheet.
  • Do not use incoming cell lines until testing has confirmed the absence of mycoplasma and the identity of the line.
  • Document new cell-line details upon acquisition. Record how many times a cell line has been passaged, as some cell lines exhibit different characteristics after multiple passages. Implement a standard for when to discard cells in culture and thaw a vial of stock.


Cell-line contamination poses a serious threat to the integrity of biomedical research. Following best practices for the validation, storage, and handling of cell culture can help to address this ongoing challenge and thereby improve the reliability of experimental research data.


Patrick Schneider, Ph.D., is head of R&D, and business development, MilliporeSigma

1. Masters JR. Cell-line authentication: End the scandal of false cell lines. Nature. 2012. 492: 186. doi:10.1038/492186a.
Nature. 1968. 217: 750–751.
3. Burnett E, Penn L, Finley D. Contaminated cell lines are bad for your health. Biofiles. 2012. 8(16).
4. Boonstra JJ, et al. Verification and unmasking of widely used human esophageal adenocarcinoma cell lines. J. Natl. Cancer Inst. 2010. 102: 271–274. doi:10.1093/jnci/djp499.
5. Razin, S. “Mycoplasmas,” in Medical Microbiology. Baron S., editor. 4th edit. 1996. University of Texas Medical Branch at Galveston. Galveston, TX.