Cell Line Misidentification and Contamination

One of the most overlooked problems in regards to cell cultures and the reliability of the results obtained from them is the issue of misidentification resulting from contamination. It is a well-known issue and easily leads to false results built upon false results:

Consequences of cell culture contamination

“Contaminants can affect all cell characteristics (e.g. growth, metabolism, and morphology) and contribute to unreliable or erroneous experimental results. Cell culture contamination will likely create a need for experiments to be repeated, resulting in frustrating time delays and costly reagent wastage. Data derived from undetected contaminated cultures can end up published in scientific journals, allowing others to build hypotheses from dubious results. The pervasiveness of cross-contaminated and misidentified cell lines is a decades-long issue; in 1967, cell lines thought to be derived from various tissues were shown to be HeLa cells, a human cervical adenocarcinoma cell line. However, studies involving these misidentified cell lines continued to feature in hundreds of citations during the early 2000s.

This pattern is a well-acknowledged problem and threatens to undermine scientific integrity. The first published retraction in Nature Methods was due to cell line contamination3, and one conservative estimate of “contaminated” literature in 2017 found 32,755 articles reporting on research with misidentified cells.4 While many scientists may have been blissfully ignorant in the past, awareness of misidentified cell lines is growing.”

“But what should be done about existing contaminated literature? Mass retraction of affected articles may disproportionately punish the careers of a few scientists, and could be a waste of resources containing potentially valuable data. One recently proposed system of “self-retraction” recommends replacing blame with praise in order to encourage self-correction.5 Post hoc labeling of published articles in the form of an “expression of concern” allows existing findings to remain accessible, while giving readers a chance to form their own judgement.”


It is obvious cell misidentification and contamination has been an oft ignored problem even though there are numerous ways in which these cell lines and cultures can be corrupted. Many of these problems were outlined in 2014 in the Guidelines for the use of cell lines in biomedical research:

4.1. Cell line misidentification

“One of the most serious and persistent problems is cell line misidentification often resulting from cross-contamination. This means that authentication is required on receipt, before storage and distribution, and after completion of a project (see Sections 1.2.2 and 1.5.1).

4.2. Mycoplasma contamination

Contamination of cell cultures with mycoplasma was first noted in the 1950s but is still regrettably often disregarded. The following important points should be noted:

  1. Mycoplasma contamination is very frequent, worldwide.
  2. Using mycoplasma-contaminated cells can result in erroneous, misleading or false experimental results.
  3. Owing to lack of visible signs mycoplasma-positive cell cultures can go unnoticed.
  4. Be aware of potential sources of mycoplasma contamination (see Section 4.2.2).
  5. Use good aseptic technique and laboratory practices to avoid mycoplasma contamination.
  6. Have an effective quarantine procedure for all untested cell lines.
  7. Establish a regular and continuous mycoplasma-testing programme.
  8. Scientific journals are starting to ask for evidence of mycoplasma testing before accepting papers for publication.

Mycoplasmas and the related Acholeplasmas (collectively referred to as ‘mollicutes’) are the smallest and simplest self-replicating bacteria and are significant in that they have become probably the most prevalent and serious microbial contaminant of cell culture systems used in research and industry today. Owing to the absence of any visible morphological changes or other symptoms mycoplasma infection of cell cultures often goes undetected. However, it is the invisible effects of the contamination on the infected cells that makes it such a serious problem. It is therefore essential that routine mycoplasma testing is performed regularly on all research cell lines to ensure the validity of study results before publication. Although >20 different species of mycoplasma have been isolated from cell cultures, >95% of infections are caused by six prevalent species, which are the following: M. argininiM. fermentansM. hominisM.hyorhinisM. orale and Acholeplasma laidlawii.

Although primary cell cultures and early passages are less frequently contaminated with reported incidences of between 1 and 5%; continuous cell lines have much higher incidences of between 15 to 35% (Drexler and Uphoff, 2002).

Mycoplasma are unaffected by many of the antibiotics commonly used in cell culture, such as penicillin and can grow to extremely high titres (typically 1 × 107 to 1 × 108 organisms per ml) in mammalian cell cultures without producing any turbidity in the medium, or other obvious symptoms. In addition mycoplasma are extremely small (0.15–0.3 μm) and pleomorphic, and will pass through standard 0.22-μm bacteriological filters (0.1-μm filters are required for sterilisation). The only assured way of detecting mycoplasma contamination is regular testing.

4.2.1. Effects

The effects of mycoplasma contamination on the host eukaryotic cell are quite variable but have been shown to alter many host cell functions including growth, morphology, metabolism, the genome and antigenicity (Drexler and Uphoff, 2002). Using mycoplasma-contaminated cultures in experiments will therefore clearly call into question the validity and significance of any research data generated and could result in the publication of erroneous experimental results. Research journals are now starting to ask for evidence that mycoplasma-free cell cultures are used in studies before accepting papers for publication. In addition the time and cost involved in cleaning contaminated laboratories, obtaining new cell cultures and repeating experiments is significant as is the potential reputational damage of publishing erroneous results.

4.2.2. Sources

Common sources of mycoplasma contamination in the laboratory include:

  1. Cross-contamination from other mycoplasma-positive cell cultures.
  2. Laboratory equipment and work surfaces.
  3. Laboratory personnel (often via respiratory tract infections).
  4. Cell culture media, sera and reagents.
  5. The liquid phase of LN2 cryostorage vessels.
  6. Feeder cell cultures.
  7. Laboratory animals

4.3. Contamination by other microorganisms

“With correct working practice it should not be necessary to use antibiotics to control contamination in established cell lines and their use should be discouraged. Microbial contamination may be obvious, indicating that the culture should be discarded, but, if antibiotics are used, contamination may be repressed but not eliminated. Such cryptic contamination may co-exist with the cell culture and only appear when the culture conditions change or the organism develops antibiotic resistance. In addition as antibiotics and antifungal agents act by inhibiting biochemical functions of the organism, these activities may also affect animal cells prejudicing the outcome of experiments. For example, amphotericin B is a membrane active agent and may therefore interfere with any mammalian cell experiments involving membrane trafficking or intercellular signalling.

4.3.1. Bacteria and fungi

If cells are cultured in antibiotic-free media as recommended, contamination by bacteria, yeast or fungi can usually be detected by an increase in turbidity of the medium and/or a change in pH (typically acidic with many bacteria giving a yellow colour change in media containing phenol red as a pH indicator but can be alkaline, pink, with some fungi). It is recommended that cells are inspected daily and must always be examined under an inverted phase microscope before use in an experiment.”

“If a cell culture is contaminated with bacteria or fungi, then the best method of elimination is to discard the culture and initiate fresh cultures from frozen stock. In the case of irreplaceable stocks, it may be necessary to use antibiotics; the more antibiotics that are tested, the greater the chance of finding one that eliminates the infection. However, if the cells have been routinely grown in media supplemented with antibiotics (which is not recommended), it is almost certain that the contamination will be with organisms that are already resistant to this and some other antibiotics.”

4.3.2. Viruses

“As long as cell culture reagents of biological origin are used, such as serum to supplement media and natural trypsin for subculture, there will always be a risk that endogenous infections in the source of the reagent will infect the culture. Any viral contaminant that grows in the cells will affect the cells’ metabolism and could also present a safety hazard to lab workers. The source of viral contamination can be from the tissue from which the cells are derived (e.g., HIV from Kaposi’s sarcoma cells, EBV from lymphoma cells). Alternatively, contamination can be derived from other infected cultures or, as a more remote possibility, from laboratory personnel. Another route of infection can be during passage of cells in experimental animals, important when considering the use of cell lines for or from implantation of xenograft tumours. Not only do the cells to be implanted need to be free from contamination by extraneous viruses but also the animals into which the transplant is to be made should not harbour viruses that could affect the growth and response to therapy of the cells under study.

Even more than with mycoplasma, elimination of viral contamination is difficult and is likely to be impossible. However, what is worse, there are no simple universal diagnostic tests to identify viral contamination. Next-generation sequencing techniques potentially offer such screening but are yet to be qualified for routine safety testing. Identifying viruses currently necessitates screening with a wide panel of immunological or molecular probes and may be best done by a specialist testing service. As yet, such testing is largely restricted to human pathogens such as EBV, HIV, HTLV I/II and Hepatitis B & C, and few laboratories screen for animal viruses on a routine basis, although some commercial suppliers and veterinary laboratories do. Use of serum-free medium and recombinant trypsin should help to minimise viral infection from reagents and GCCP will minimise the risk of transmission from one culture to another or to the person handling that culture.

4.3.3. Prions

Transmissible spongiform encephalopathy (TSE), including what is known as bovine spongiform encephalopathy, BSE, or mad cow disease, is unlikely to be present in cancer cells or tissue culture products. Risks of prion contamination may need to be considered when using cell lines from the CNS or from patients with certain diseases associated with abnormal prion expansion. It should be noted that prions are not destroyed by autoclaving or by most chemical disinfectants. Disposal into 10% hypochlorite followed by incineration is recommended for any contaminated material.

4.4. Genetic instability and phenotypic drift

Two other major problems that can affect the utility of cell lines are genetic instability and phenotypic drift, both of which may progress the longer the cell line is cultured. Records should be kept of the length of time a cell line has been kept in culture. For finite cell lines, this is determined by the generation number, the number of doublings since isolation (necessarily approximate as it is difficult to measure the number of doublings in the primary culture). This number will determine the lifespan of the culture as most finite cell lines will die out due to senescence at between 20 and 60 doublings, which means that they can only be used reproducibly between 15 and 45 generations depending on the cell type. Cells are frozen at the lowest generation number possible and used to replace stocks at regular intervals before the onset of senescence. When thawed the generation number resumes at one over the number at freezing. For continuous cell lines, the number of passages since last thawed from the freezer is recorded. If the passage level at freezing is known, this may be added on but often this is not known.

4.4.1. Genetic instability

The chromosomal content of most continuous cell lines is both aneuploid (abnormal chromosome content) and heteroploid (variable chromosome content within the population). Many cancer cell lines have defects in p53 and other genes that monitor and repair DNA damage, resulting in an increased mutation frequency. Hence, the genotype of continuous cell lines can change with time and cell lines should not therefore be maintained for extended periods of time in continuous culture (Wenger et al, 2004Saito et al, 2011).”

4.4.2. Phenotypic instability

“Lack of expression of the differentiated properties of the cells of origin is a major recurrent problem. This can be due to selection of the wrong cell lineage in inappropriate culture conditions. For example, a disaggregated skin biopsy will ultimately give rise to a fibroblastic population that overgrows the epidermal keratinocytes, unless selective conditions are used. However, even under selective conditions, the need for propagation stimulates cell proliferation rather than differentiation. This process can either select undifferentiated cells or can lead to a loss of differentiated characteristics. In some cases, such as fibroblasts or endothelial cells, this is due to dedifferentiation, but in others, such as mammary epithelium, it is probably due to propagation and expansion of the progenitor cell compartment, which lacks the differentiated characteristics.

4.4.3. Stability of stem cell lines

“There are particular problems associated with cell lines derived from stem cells, whether embryonic, fetal, adult or iPSCs.”

“Hence, different conditions need to be defined for culture of a cell line dependent on whether cell proliferation or cell differentiation is required. It is important and probably essential for comparative purposes that different laboratories using the same cell line should match their culture conditions as closely as possible.”


Cell line misidentification due to contamination from mycoplasma alone can cause alterations to many host cell functions such as growth, metabolism, antigenicity, morphology, and changes to the genome. Other “viruses,” antibiotics, parasites, and amoebas are also said to be sources of contamination which can effect the outcome of the cell culture experiment in various ways. Even the length of time the cells are cultured can effect genetic and phenotypic stability.

Needless to say, there are numerous ways in which cell lines are misidentified and contaminated. It is impossible to say that the cell culture is free of all contaminants as many are unknown or are unable to be detected. The means by which cells are decontaminated with antibiotics can have disastrous effects on the cell culture.

There is no way to state that the results and evidence from cell cultures are reliable for various reasons (cells/chemicals/antibiotics used, lack of reproducibility, inability to recreate in vivo environment). High up on that list is the well-known and often ignored issue of misidentification/contamination.

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