Authentication of cell lines: ignore at your peril!

Data presented in 1966 at the Second Decennial Review Conference of the American Tissue Culture Association [1] indicated that many cell lines currently in use in cancer research were not what they were supposed to be but were, in fact, HeLa cells. This finding was received by some with consternation, by others with disbelief. After 10 years, Nelson-Rees and colleagues were still trying to convince the scientific community that a problem existed, but sadly the evidence fell mostly on deaf ears [2]. Now, 40 years later, there is an increasing volume of research based on misidentified cell lines (over 100 misidentified cell lines have been reported) but alerting the scientific community to the problem is still incredibly difficult.

At the time the evidence for cross-contamination and misidentification first became apparent its demonstration required chromosomal and isoenzyme analysis of cell lines, techniques that were quite laborious and not always available in the average tissue culture laboratory. Now, DNA profiling has simplified the procedure considerably [3], is in widespread use and, where it is not, is available commercially for a modest sum. So why do problems of cell-line misidentification not only persist but continue to arise?

It is well established that a significant number of cell lines in current use are not what those using them believe them to be. KB cells, often quoted as examples of oro-laryngeal squamous carcinoma [4], are actually HeLa cells (cervical adenocarcinoma) and have been known to be HeLa cells for 40 years [1]; they have been cited 525 times in the past 5 years. Hep-2 cells are not hepatocellular carcinoma but are also HeLa cells [1]; they have been cited 667 times in the past 5 years. MCF-7/AdrR cells are not a drug resistant derivative of MCF-7 breast carcinoma cells as claimed [5], but are actually OVCAR-8 ovarian carcinoma cells; they have been cited approximately 300 times in publications on drug resistance in breast carcinoma [6]. If there are over 100 misidentified cell lines that have been detected, one must assume that the number being worked on but never achieving publication will be far greater.

Estimates of the incidence of cross-contamination or misidentification vary but it is probable that the interpretation, with regard to tissue or cell-type specificity, of between a tenth and a third of experiments carried out with cell lines, may be invalid because of the false identity of the cells [7,8]. The waste of time and resources that this confusion implies is substantial. Not only are there many studies that are invalid before they start, but also many others that are successfully published and lead to the dissemination and general acceptance of erroneous data. A few years ago, a new cell line appeared [9] and seemed to satisfy the needs of the large number of people working in vascular research and angiogenesis. This line was ECV304, supposedly a continuous endothelial cell line, but was shown in 1999 to be T24 bladder carcinoma [10]. Even so, it is still being used as an endothelial cell line [11] and 35 articles have been published in the past year.

It has been claimed that there are many types of work employing cell lines where the actual identity of the cell lines is unimportant. This may be true and the results valid, even if the cell line is misidentified; however, the problem arises when this work is then cited by someone else who wrongly attributes the results to a specific type of cell. For example, the demonstration of the mechanism of multidrug resistance in KB cells [12] is perfectly valid but the deduction from this that multidrug resistance applies to oral epidermoid carcinoma is not [13].

Cross-contamination or misidentification arises due to the following:

•	Poor technique when handling cell lines, allowing cells from one cell line to be introduced into the medium or culture vessel of another cell line. If the growth rate of the contaminating cells is faster they will overgrow and eventually replace the slower growing cell line completely;
•	Mislabelling of culture flasks;
•	Poor management of liquid nitrogen storage (poor labeling and inventory control) resulting in one vial of cells being thawed erroneously in place of another;
•	Overenthusiastic claims from originators of cell lines excited by the development of a new cell line overshadows adequate authentication [14];
•	Passing cell lines ‘down the line’ from one laboratory to another without proper authentication by the recipient.

Misidentification and cross-contamination still occur because of a general lack of appreciation of the scale of the problem among the scientific community. At one time, cell culture was a specialised discipline limited to a small number of enthusiasts familiar with its history and problems. Now it is a routine tool used by many who are not interested in cell culture for its own sake, but merely use it as a tool. This in itself is not wrong; it is what many people in the field have been promoting for years, but what is wrong is that many incomers to the field do not have adequate training. While the biotechnology industry has taken on board the clear necessity for quality assurance (QA), many independent and academic research institutes lack clarity in establishing responsibility for QA at the individual, departmental and institutional level. Most scientists appreciate the need for the assurance of the purity and integrity of chemicals that they use – why is this not applied more rigorously to cell lines?

There are a number of steps that can be taken to prevent cross-contamination. When a new cell line is started in a laboratory, steps must be taken to ensure that the cell line is comprised of cells of the same origin as those used to initiate the culture. The best way to achieve this goal is to retain tissue or DNA from the original donor and perform DNA profiling when a cell line becomes established, before it is frozen, when it is thawed and before it is passed on to another user. Currently, DNA profiling applies mostly to human and murine cell lines, but primers are becoming available for other species. Another option is to target mitochondrial DNA where discrimination among 45 species may be possible [15].

Existing cell lines should be sourced from a reputable cell bank, such as the American Type Culture Collection [101], the European Collection of Cell Lines [102], Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (German Collection of Microorganisms and Cell Cultures) [103] or the Japanese Collection of Research Bioresources [104], who will have carried out the necessary authentication. Cancer Research UK also maintains an authenticated bank of around 2000 cancer cell lines available to grant holders. Even if you do not obtain cell lines from one of these sources, accessing their online catalogs will provide information on the authenticity of many of the commonly used cell lines and will indicate clearly if they have been misidentified or cross-contaminated. Cell lines that are obtained from other sources must be authenticated, even if obtained from the originator.

A number of strict procedures are essential to maintain the asepsis and integrity of a cultured cell line [16]:

•	Use a separate medium and other reagents, such as trypsin, for each cell line;
•	Do not have culture flasks and media and reagents bottles for more than one cell line open at the same time;
•	Handle rapidly growing cell lines, such as HeLa, on their own and always after handling other cell lines;
•	Never use the same pipette for different cell lines;
•	Do not put a pipette back into a bottle of medium after it has been in a culture flask containing cells;
•	Always use plugged pipettes;
•	Do not use pipettors for routine maintenance unless the tips are plugged;
•	Check the appearance of the culture before and after every subculture and compare with standard reference photographs;
•	Confirm the identity of a cell line before cryopreservation and after thawing from storage.

Action is required at a number of different levels. The prime responsibility lies with institutional directors and those directly supervising research based on the use of cell culture. Supervisors must ensure that their staff are properly trained and made aware of the risks of cell-line cross-contamination and misidentification [3,8,17–19] and institutional directors or heads of departments have the responsibility to make sure that their senior staff are properly informed. Unfortunately, it must be accepted that many people in a supervisory role are not always aware of the problem and its severity, partly because many reports of cell-line cross-contamination appear in specialist tissue culture journals rather than in mainline cell and molecular biology and cancer journals (‘preaching to the converted’), so other steps are required. There is clearly a need for ready access to a list of suspect cell lines, perhaps on a website with links from cell banks, relevant scientific societies, journal instructions to authors and granting agencies’ instructions to applicants and awardees. Currently, this author maintains such a list and hopes to make it generally available in the near future.

Scientists now accept that work with human material must be cleared with a local human ethics committee, and work with animals, similarly, should be cleared by a local animal ethics committee. It does not seem unreasonable to require that proposals for projects with cultured cell lines should be similarly regulated and adhere to specified QA standards, such as authentication and freedom from contamination. In a small research group, this may simply require the appointment of a responsible and informed individual, but a larger institution may require a more formal QA standards committee, whose remit might include PCR primers and plasmids, as well as cell lines.

Most research requires outside funding so granting agencies have a critical role to play; without funds the work cannot proceed. While it may not be feasible for granting agencies to police the practices of research institutes, they can bring pressure to bear if the instructions for grant applicants and for grant reviewers insist that applicants provide evidence (their own or from a reputable external agency) that confirms the authenticity of any cultured cell lines that they propose to use, and if the terms and conditions of funding state that if evidence emerges during the work that cell lines in use have not been properly authenticated, funding may be withdrawn.

Although it would be preferable to prevent work with false cell lines from starting, it is inevitable, given the experience of the past 40 years, that some projects will reach the stage of publication. Hence there must be another control at this level when the work is likely to be accepted by the readership of the journal, given that it has passed peer review, and propagated by future citations as relevant to a particular cell type. Instructions to authors should include a requirement for the proof of authenticity of cell lines used in the work and reviewers of manuscripts must be instructed to return manuscripts that do not contain proof of the identity of cell lines and reject them if this evidence is still lacking in the revised manuscript.

Given the adoption of cell culture techniques in the biotechnology industry, the concept of quality assurance is an important ideal to instil into students. It is vital to include a lecture, or component of a lecture, dealing with problems of contamination and authentication in any undergraduate or graduate courses which teaches cell culture, even as a minor part of another program.

Problems of cross-contamination have been evident in work with cultured cell lines for over four decades. With the identification techniques that are available now there is no excuse for it to continue at its present unacceptable level. It is probably fair to say that most erroneous use of cell lines is not intentional and there is no attempt at scientific fraud; the cause is ignorance or, at least, a lack of awareness in scientists conducting research. This results in misinterpretation of experimental results both by those conducting the experiments and by those who subsequently read the published work. It is the duty of all who are aware of the problem to make sure that information regarding the widespread occurrence of misidentification and cross-contamination is disseminated and comes to the attention of those who most need to know, in other words, those culturing the cells and those who supervise them. It appears it will not happen by simple diffusion of knowledge but will require positive action such as improved teaching at the graduate and undergraduate level and the institution of restrictions on funding and publication. New misidentified cell lines will continue to arise, but, with the proper practices in place, it should be possible to eradicate the problem completely.

Financial & competing interests disclosure

The author has no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending or royalties.

No writing assistance was utilized in the production of this manuscript.