Verifying cell lines: Challenges and best practices

Screen Shot 2017-05-11 at 5.32.18 PM Michelle Newman

Ever since the isolation and successful culturing of HeLa cells, working with cell lines has been and will continue to be an important part of the scientific process. We now have hundreds of cell lines from a multitude of cancers, tissues, diseases, and species. But despite the many research possibilities they enable, these various cell lines are not without their challenges. Some of these pitfalls are outlined below, followed by solutions and best practices.


Culturing cells can be problematic. Firstly, there is the issue with long-term sub-culturing. It has been reported that continuous passaging results in a change to cell line traits induces senescence and can introduce genotype instability, as well as new mutations that alter cell phenotype.

One of the factors contributing to cells being cultured too long is tracking the passage number. There is no set rule for when to “retire” a sub-culture, and the time frame varies from cell line to cell line, tissue of origin, and species. Further problems depend on how we number the passages. For example, some will record the passage number as 1 when the cells are thawed, while others will continue with the passage numbering as at the time of freezing. I have also witnessed people passaging cells without ever recording a passage number! These differences in laboratory practice can obviously affect the time for which a stock is sub-cultured.

Another common issue is inadvertent colony selection from over-zealous passaging. This means “splitting” the flask of cells at a dilution that is too high, resulting in the selection of a sub-population of cells, or cells that are not in exponential growth that can lead to senescence.

Cross-contamination and mislabelling

Due to the relatively low cost of cell lines and associated reagents, labs can culture multiple cell lines simultaneously—often from different species. There are also individual differences in culturing and aseptic technique, as well as the commonplace donation of cell lines between colleagues. This has led to cross-contamination and mislabelling.

Cross-contamination of cell lines or working with a cell line that has been misidentified can lead to wasted research expenses and unrepeatable experiments. Retraction Watch has reported multiple examples of articles retracted due to misidentified cell lines (for example), and in 2015, Nature and its affiliated journals announced that cell lines used in experiments need to be verified, including the source of the cells.

Like most researchers, I would be very frustrated to find out that the lung cancer cells I had been working with were, in fact, spleen cells!

Picture1 credit: Ed Himelblau,

Due to the obvious dilemma of publishing data or trying to repeat experiments with misidentified or cross-contaminated cell lines, there are many ways in which a researcher can verify their stocks. The most common, and only industry standard test, is the short tandem repeat (STR) profiling—a technique used in forensic testing because it can be used on different platforms, and can detect DNA mixtures.

The eight core STR loci will show relatedness between cell lines by amplifying a unique DNA pattern of repeat units and interrogation of a Y-chromosome gene. Since the most common contaminating cell line is HeLa, a HeLa marker chromosome can be included, while isoenzyme analysis is used to identify species.

There are presently 488 cells labelled as misidentified or cross-contaminated. The full list can be found at the International Database for Cell Line Authentication, and the ATCC lists cell lines from its stock that have been found to be cross-contaminated or misidentified.


The ATCC has a number of guidelines for culturing cells, as do companies who sell cell culture reagents. Here is an overview of recommendations:

  • Note all details on vials when cells are frozen: date, cell line, passage number, who froze the cells, and number of cells.
  • Continue with the passage numbering of thawed cells as stated on the vial.
  • Split cells in the exponential phase (for a T75 this is usually every two days at a 1/10 or 1-2 million cells dilution).
  • Change media regularly.
  • Thaw a new aliquot of cells after a maximum of three months of culturing cells.
  • Discard cells if growth slows or morphology changes.
  • Only passage one cell line at a time and decontaminate the biosafety hood in between cell lines.
  • Maintain dedicated aliquots of media for each cell line.
  • Empty waste containers regularly.
  • Maintain a lab-wide standard operating procedure.
  • Verify all donated cell lines.
  • Regularly screen cell lines for cross-contamination.

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Michelle Newman

Michelle Newman

Michelle’s PhD focus was to understand the role of DNA methylation in the radioadaptive response and has since taken an interest in RNA processing and DNA damage, and has a strong molecular biology background. When she isn’t writing on her blog, she can be seen weaving through the streets on her bicycle!