This story has been updated to clarify the collaboration between NIST and ATCC.
NEW YORK (GenomeWeb) – The National Institute of Standards and Technology (NIST) has developed a method to identify cell contamination in mouse cell lines, the first step in addressing an issue that has plagued research labs for years.
The method involves using 19 short tandem repeat (STR) markers to identify cell lines through a highly multiplexable PCR-based assay. The institute developed the markers and the assay, and are validating their markers through a collaboration with ATCC, a private nonprofit standards organization that also serves as a biological cell repository.
Cell line misidentification is an issue that affects many research laboratories and has been pointed out in numerous peer-reviewed publications over the last decade. According to ATCC researcher and consultant Yvonne Reid, misidentification affects up to an estimated 15 percent of the most frequently used cell lines in human cell lines alone.
"The misidentification of cell lines has been known for a long time, going back to the 60s, and maybe before," Reid said in an interview. "A lot of the misidentification that we have heard about pertains to human cell lines."
In human cell lines, misidentification or contamination of research has created issues revolving around study irreproducibility which in turn leads to added research costs once scientists try to correct the issue retroactively. STR-based cell line authentication methods have already been established for human cell lines, through the ATCC and other prominent organizations. "[Human cell line] STR markers are very well characterized in the forensics community, and really the cell community just borrowed that technology and then wrote standards for it," Jamie Almeida biologist at NIST and lead researcher on the ATCC collaboration, said.
But while human cell lines are the most commonly used cell lines in the scientific research community today, this issue is not species specific.
"I think there is some indication, especially from the [International Cell Line Authentication Committee] misidentified cell lines, that … a minimum of 10 percent of non-human cell lines have been misidentified," Maryellen de Mars, senior director of the ATCC's Standard Research Center, said
The NIH is one of several organizations that have made policy changes over the past few years to enforce the need for cell line authentication in research, which includes determining the cell line's identity and establishing that it contains no microbial contamination. Last January, the NIH officially changed some of its grant application requirements. The organization now requires that researchers provide a plan to authenticate all biological and chemical reagents, and if it doesn't meet peer review the application will not get funded until the issue is adequately addressed, said Jon Lorsch, director of the NIH's National Institute of General Medical Sciences.
The importance of enforcing cell line authentication is especially significant in mouse cell lines since the NIH regards the mouse as a model for different disease, especially cancer, Reid said. It's critical to ensure that researchers catch misidentification and contamination issues in this type of research to ensure that it doesn't have downstream consequences for drug and therapy development, Lorsch added.
Efforts by the NIH, other funding agencies, and publishers to recognize that misidentification of cell lines leads to irreproducibility in science has helped to encourage the development of better authentication technology, de Mars said.
The mouse cell line identification method developed by NIST and ATCC requires a standard thermal cycler to amplify the STR target regions within the DNA, and the NIST team has used the Thermo Fisher Scientfic Applied Biosystems Veriti thermal cycler, Almeida said. "Then to separate your PCR products you have to run capillary electrophoresis. We currently use the ABI 3500 XL, which is a 24-well capillary instrument," she added.
In its current form, the assay uses 19 STR markers that researchers believe establish clear distinctions between mouse cell lines, even those derived from the same inbred mouse strain, Almeida said.
NIST was recently granted a US patent covering the first nine STR markers that were initially identified. "We started out initially with nine markers, but we found that was not enough to discriminate among the various cell lines that the [ATCC] provided," Reid said. Consequently, the researchers worked to develop additional markers and ended up with their final list of 19 that all the researchers feel are "robust" enough, she added. NIST has since submitted a second patent application to cover the 10 additional markers that make up the final assay.
"The patent that's in progress now for the additional STR markers, defines the primer concentrations, and how to put the whole assay together," Ken Cole, group leader of the Bioassay Methods group at NIST, said. "It includes data about the sequence of the STR markers but the key is how to put [the markers and primers] all together and make them work with each other."
However there was a great deal of work put in before the researchers arrived at their current assay. Most of the work went towards ensuring that there was a sufficient number of STR markers that work with each other, and developing primers to establish significant cell line differentiation, Cole added.
Establishing these STR markers for a functioning assay is the first step in ongoing research on mouse cell line authentication. The next step is to develop a cell line database, de Mars said.
This database will be made up of STR markers for the approximately 50 mouse cell lines that ATCC has donated. The ATCC researchers hope that the database not only provides important information to the research community, but also put in place a platform other researchers could use to upload additional data, Reid said.
Once all of the testing and database infrastructure is in place, ATCC plans to create a set of consensus standard, which it has already done for STR-based cell line authentication in human cell lines, for mouse cell lines.
The STR marker-based method holds promise because it has the ability to correctly identify different mouse cell lines at a higher resolution than previous methods.
But STR-based authentication doesn't offer a perfect solution. It requires a lot of time and effort on the front end to identify the STR markers of interest in each cell line and create an assay that is robust enough to accurately identify closely related cell lines. "We are very interested in promoting additional sorts of technologies to add to the armamentarium of approaches," Lorsch said. "For instance SNP … approaches would be a useful, somewhat orthogonal method. The more kinds of approaches we have the better our resolution will be."
"I would not say that we have solved the problem yet, but I think we have taken a very significant step in requiring these authentication plans, both in terms of actually getting people to authenticate the cell but also in raising awareness in the entire community of the seriousness of the problem," Lorsch said.