Cell line misidentification is both a devastating and frighteningly common problem in life science research. One report on the scope of the problem identified 32,755 scientific papers reporting on research using misidentified cells, which were further cited by an estimated 500,000+ more papers.1 According to some reports, as many as one-fifth to one-third of all cell lines may be misidentified or contaminated. The potential consequences of this problem, which include failed experiments and retracted papers – equating to significant losses in time, funds and reputation – should motivate all laboratories and researchers to take every measure to prevent and detect cell line misidentification. Here are some tips to help biological researchers stay vigilant and ensure the integrity of their cell culture experiments:
1. Be mindful when sourcing cells
When purchasing or receiving a new cell line, you should be able to have confidence that the cells coming into your lab are correctly identified. Before selecting any cell line to work with, researchers should consult the Register of Misidentified Cell Lines maintained by the International Cell Line Authentication Committee (ICLAC).2 Cellosaurus is another useful resource for selecting suitable cells and researching “problematic” cell lines when planning experiments.
Any new cells should always be acquired from a reputable source, such as an established cell repository, or directly from the laboratory where the cell line was created. This is the best way to ensure that the cells are authenticated and correspond to the original donor. Reputable sources should provide documentation of cell line authentication, which the receiving lab should keep for their records to ensure traceability. Cells received from secondary sources, such as a lab other than that which established the cell line, are more likely to be misidentified or contaminated than those from a primary source or reputable cell repository.
2. Use genotype-based test methods to authenticate cells
Even when receiving cells from a reputable source, it is best practice to perform your own authentication testing prior to establishing a cell bank or starting a new research project. Authentication testing should also be performed after experimentation, prior to submitting any results for publication, to rule out any contamination that may have occurred during the experimentation period.2 Genotype-based methods are far more reliable for authenticating cell lines than phenotype-based methods, although phenotype changes – such as changes in growth rate or differences in morphology viewed under a microscope – can be indicators of possible contamination or genetic drift that should be investigated with genetic testing.
Short tandem repeat (STR) genotyping is the gold standard method for authentication testing of human cell lines, and STR methods have also been developed for some other species, such as mice, rats and African green monkeys.3 This method involves the amplification of STR loci via polymerase chain reaction (PCR) and determination of STR allele lengths via capillary electrophoresis. The STR allele data acquired from the cells can be compared to reference databases to confirm whether the cell line tested matches with the original cell line or donor tissue. STR genotyping has excellent discriminating power and thus can provide high confidence that your cell line is authentic, as well as reliably detect misidentified cell lines. If an STR-based method is not available for your cell line species, an alternative genotype-based method validated for the species should be used, such as a method based on DNA “barcode” sequencing.2
3. Follow best practices to avoid contamination during handling
Cell cultures may be contaminated by microbes such as bacteria or fungi, or may be cross-contaminated with other cell lines. While it is important to prevent both types of contamination, cross-contamination between cell lines may go undetected due to similarities between cells of the same species or from similar groups (i.e. mammalian cells), allowing the contaminated cell line to perpetuate. Contamination can be prevented by following good cell culture handling practices, including frequent, thorough sterilization of workstations and materials, and isolation of different cell lines from one another.
All surfaces and equipment that will be used when handling a cell culture should be thoroughly cleaned with a disinfectant such as 70% ethanol, including gloves, incubators, microscopes, reagent bottles, etc.4 Autoclaving can also be used to decontaminate items such as incubator racks or autoclavable pipettes. Any tasks or experiments to be carried out in a biosafety cabinet should be planned ahead of time so that only the necessary items are brought into the hood. Clutter inside the cabinet can disrupt the airflow, and the risk of contamination is lessened when items are spaced apart and well-organized to reduce unnecessary arm movements inside the hood. Bottles should be capped when not in use, and materials to be pipetted should be poured into a sterile container rather than pipetted directly from the stock solution, which risks contaminating the entire stock.5 Additionally, it is important to give the hood enough time to establish a clean air flow – wait at least 15 minutes after turning it on before beginning your work.
To prevent cross-contamination between cell lines, it is essential to work with only one cell line at a time. Different cell lines should not be brought into the same workspace, and cleaning procedures should be repeated when switching work to another cell line. Additionally, media and reagent supplies should be dedicated to specific cell lines to further ensure isolation of different cell lines from one another.
4. Maintain accurate and thorough documentation and labeling
Proper documentation and labeling of cell lines can prevent many problems that may lead to misidentification, such as mislabeling of containers or gaps in information about cell origin, phenotype or authentication. Laboratories should keep clear, accessible records about the origins of each cell line, including donor and disease information, cell type, when the cell line was received and where it was acquired from.2 Additionally, researchers should regularly document information on cell line handling, tests performed (including authentication tests), passage numbers and phenotypic observations, which can include microscope images and growth data. This documentation can help identify any changes that may indicate contamination, demonstrate when the cell line was last authenticated and help trace backward along the chain of handling when problems arise.
All cell receptacles, such as cryovials, plates or flasks should be accurately and carefully labeled, using care to ensure that the label is readable and firmly affixed to the container. Printed labels are often preferable as they remove readability issues that can arise from handwriting, are less prone to smudging and are also more efficient to produce. Printed labels also enable the use of barcoding to help document cell line handling. Labels should be written or printed using solvent-proof ink, and containers that will be placed in a freezer or cryogenic storage should be affixed with labels designed to withstand low temperatures, as normal adhesives may fail in these conditions. The label should include sufficient information to identify the cell line or culture, such as the cell line name, date, batch number, passage number, etc.
5. Dispose of any compromised cells
Unfortunately, once a cell culture, batch of cultures or cell line stock is contaminated or otherwise compromised, the cells should be disposed of – it is rarely worthwhile to attempt to recover a partially-contaminated cell culture or stock, even when some of the original cell line is present, nor is it worth the risk to use a cell line that is missing a label or is known to have been exposed to cross-contamination. Fortunately, laboratories can minimize the impact of cells that have been compromised due to contamination, genetic drift, or otherwise, by maintaining a tiered cell bank system that preserves a supply of early passage, authenticated cell stock.
A master cell bank should be established whenever a new cell line is acquired, with the cell line first held in quarantine until it can be tested for mycoplasma and authenticity.6 Once the cell line is authenticated, it can be cultured and expanded to produce a master bank that is stored and preserved in cryogenic conditions. A small portion of cells from this master bank can be thawed and expanded to produce a working bank, with additional quality and authentication testing performed prior to this expansion. Lastly, cells from the cryopreserved working bank can be thawed and cultured for research projects after an additional QC and authentication check. Compromised cells can be replenished from the working bank, and the working bank can be replenished from the master bank when needed. It is advisable to store the master bank and working bank separately in case a major incident, such as a power outage, compromises an entire stock. This tiered banking system provides multiple “back-up” options, and multiple working stocks can be made from the same master stock to supply cells for different groups or applications.
References
- Horbach SPJM, Halffman W (2017) The ghosts of HeLa: How cell line misidentification contaminates the scientific literature. PLOS ONE 12(10): e0186281. https://doi.org/10.1371/journal.pone.0186281
- "Making Cell Line Authentication Part of Everyday Cell Culture Practice," International Cell Line Authentication Committee (2023). https://iclac.org/wp-content/uploads/ICLAC_Advice-to-Scientists_02-Mar-2023.pdf
- Almeida JL, Korch CT. Authentication of Human and Mouse Cell Lines by Short Tandem Repeat (STR) DNA Genotype Analysis. 2023 Jan 17. In: Markossian S, Grossman A, Brimacombe K, et al., editors. Assay Guidance Manual [Internet]. Bethesda (MD): Eli Lilly & Company and the National Center for Advancing Translational Sciences; 2004-. Available from: https://www.ncbi.nlm.nih.gov/books/NBK144066/
- Tanabe, L. Strategies to Minimize Contamination in the Cell Culture Lab [Online]; Biocompare: 2019. https://www.biocompare.com/Application-Notes/364031-Strategies-to-Minimize-Contamination-in-the-Cell-Culture-Lab/ (Accessed March 17, 2023)
- "10 Basic tips for mammalian cell culture," Blog Post by Sana Khan Khilji, AddGene (2018). https://blog.addgene.org/10-basic-tips-for-mammalian-cell-culture
- Fundamental Techniques in Cell Culture: Laboratory Handbook 4th Edition; European Collection of Authenticated Cell Cultures/Merck KGaA: Darmstadt, Germany, 2018; pp 23-25. https://www.culturecollections.org.uk/media/161749/ecacc-lab-handbook-fourth-edition.pdf