New Resources for an Old Problem: Cell Line Cross-Contamination

In 2004, Dr Walter Nelson-Rees was awarded a Lifetime Achievement Award by SIVB for his pioneering work on cell line cross-contamination (1). A decade later, is cross-contamination still a problem? Cross-contamination occurs when a culture is accidentally contaminated by cells from another cell line. The end result is a misidentified cell line – a cell line that no longer corresponds to its original donor culture. The problem was first discovered by Stanley Gartler in 1967. Walter Nelson-Rees extended Gartler’s original work, using isoenzyme analysis and karyotypic analysis to detect cross-contamination. By the time Nelson-Rees (prematurely) retired from science in 1980, he and his colleagues concluded that more than 80 cell lines were cross-contaminated (2). The response from the scientific community was mixed. Some scientists accepted his findings, while others ignored or refuted his work. Although Nelson-Rees convinced many scientists that cell line cross-contamination was a common cell culture problem that must be addressed, his journey of discovery was a lonely road for one man to travel.
Fast forward to the present day. Many laboratories continue to uncover new cases of cell line cross-contamination, aided by the sensitivity and precision conferred by forensic DNA profiling. The problem is still frequently seen in laboratories and recent publications. Data from leukemia-lymphoma cell lines (n=620), analyzed by the German Collection of Microorganisms and Cell Cultures (DSMZ), show that 13% of these cell lines were contaminated with another cell line, of which 6% were also contaminated with mycoplasma (3, updated from Ref. 4). It follows that a substantial number of cell lines in publications that lack provenance or authentication data are likely to be false (5).

Collaboration and consensus are essential for us to develop effective solutions. Everyone involved in testing – including cell banks and testing laboratories – needs to agree on what test methods to use, and consider how best to share data with the scientific community. A Standard has now been published by the American National Standards Institute (ANSI) on authentication of human cell lines (6). The Standard concludes that short tandem repeat (STR) profiling is an effective way to authenticate human cell lines, and sets out a framework for a shared database. This framework has been adopted by the US National Center for Biotechnology Information (NCBI) for its BioSample database, which will enable all laboratories to share human cell line STR profiles (http://www.ncbi.nlm.nih.gov/biosample/).

Resources for the scientific community are important to help everyone respond to the problem of cell line cross-contamination. For example, scientists need to quickly check if a cell line is known to be cross-contaminated before they begin culture work to characterize further. A database of cross-contaminated or otherwise misidentified cell lines was developed by Amanda Capes-Davis and Ian Freshney, and first published in 2010 (2). The database currently lists 472 cell lines that are known to be cross-contaminated, with references so that scientists can look up the original data. Legitimate derivatives are excluded from the database by considering the provenance of each cell line. In 34 cases, cell lines were initially thought to be cross-contaminated but authentic stocks can be found. Those cell lines are listed separately in the database, with information on where to find authentic stocks where known.

The International Cell Line Authentication Committee (ICLAC) now curates the database of misidentified cell lines (2). ICLAC is an independent scientific committee with broad support from the scientific community. Established after publication of the Standard on human cell line authentication, ICLAC was formed as a voluntary initiative to improve awareness of misidentified cell lines. As an ongoing activity, ICLAC also develops resources for the scientific community where these are needed.

ICLAC’s resources are available from its website (www.iclac.org/resources/). Resources include guidelines for human cell line authentication, and advice for scientists on incorporating authentication testing into everyday cell culture practice. To improve reporting of cell lines, ICLAC has just released a cell line checklist for manuscripts and grant applications, and guidelines for naming a new cell line.
What comes next? As a scientific community, we need to consider how best to manage authentication of non-human cell lines. Multiple species are used in cell culture, and some of those are from highly inbred lab animals with near identical DNA profiles, which will prove challenging to authenticate using current methodologies as used with human cell lines. A new Standard is currently in development for non-human cell lines, focused on species level detection. We need to welcome new techniques for authentication testing, and consider how to incorporate those approaches going forward. And we need to develop policies that standardize what scientists, journals and funding bodies require when it comes to cell line reporting, to improve the quality of published work.

Finding a solution to the problem of cell line cross-contamination remains a challenge. But at least, with a collaborative approach to the problem, we will have company on the lonely road.

Submitted by Amanda Capes-Davis on behalf of ICLAC, info@iclac.org.

Further Reading

1. Masters JR (2004) Walter Nelson-Rees wins Lifetime Achievement Award. https://sivb.org/InVitroReport/39-1/lifetime.htm.
2. Capes-Davis A et al (2010) Check your cultures! A list of cross-contaminated or misidentified cell lines. Int J Cancer 127(1): 1-8. PMID: 20143388. For the latest version of the database and associated statistics see: http://iclac.org/databases/cross-contaminations/.
3. Capes-Davis A et al (2013) Match criteria for human cell line authentication: Where do we draw the line? Int J Cancer 132(11): 2510-9. PMID: 23136038.
4. Drexler HG et al (2002) Mix-ups and mycoplasma: the enemies within. Leuk Res 26(4): 329-33. PMID: 11839374.
5. Masters JR et al (2012) Cell-line authentication: End the scandal of false cell lines. Nature 492(7428): 186. PMID: 23235867
6. ANSI/ATCC ASN-0002-2011. Authentication of Human Cell Lines: Standardization of STR Profiling. ANSI eStandards Store, http://webstore.ansi.org/.