Misidentification of cell lines: the beginning of the end

In May 2010, the ATCC SDO (Standards Development Organization) working group published a forward-looking report entitled "Cell line misidentification: the beginning of the end" in the journal Nature review cancer. The following is the full text.

Summary:

Cell lines as a model cell of normal or tumor tissue are widely used in scientific research and drug development, but a large part of cell lines are mislabeled or replaced by cells of other individuals, tissues, and germline sources, which cannot be solved by the scientific community. Such problems have led to the publication of a large number of academic papers that are misleading or have potential errors due to the use of misidentified cell lines. The STR typing method developed by recent efforts has become a standard method for identifying human cells, and the application of STR typing has become an important step in eradicating the problem of cell misidentification.

Cell lines are widely used in biomedical research as in vitro models. The acquisition of correct data often depends on the characteristics of the cell line, especially when it is used to replace the tissue of its source. Surprisingly, the incidence of cell misidentification is not so low that it is often wrong to classify a cell as a source of another cell line. . The problem of cell misidentification has been 50 years old. Based on the analysis data of the international cell bank, the cell misidentification rate was 16% in 1977 and 18% in 1999. Until recently, cell lines used in biomedical fields need to be identified. The problem is still rarely noticed. So the members of the ATCC Standards Development Organization Working Group ASN-0002 (SDO, a working group currently developing human cell line identification standards) wrote this scientific and social article. Founded in 2007, ATCC SDO aims to develop the best standards for life sciences and to use consistent processes to balance industry, government, coordination, and academia. We expect this draft standard to be reviewed and commented by the public in 2010, and the final draft is subsequently approved by the National Institute of Standards (ANSL).

Here we describe the causes of the formation of cell misidentification, the impact and history of the scientific community, and the efforts to solve this problem. We discussed different methods currently available for cell identification and recommended the use of STR typing to identify human cell lines. Perhaps because of its importance, a generic STR classification database is under construction.

Discovery of cell misidentification

The misidentification of human and animal cell cultures is a long-standing problem that was initially recognized as dating back to the 1950s. Karyotype analysis and immunology methods were first applied to cell identification, and when a large number of germline misidentifications were reported, the ATCC was prompted to establish a cell line identification library in 1962.

Intraspecific cells were not identified in 1962, and by 1966 Stanley Gartler introduced the concept of biochemical polymorphisms to identify human cells (based on isoenzyme expression). In 1966, at the second decade of retrospectives on cell, tissue and organ culture, Gartler reported a phenomenon in which it is speculated that the cell lines derived from 18 independent individuals are all Hela cells (Hela cells are human) An established cell line, for example, consists of normal intestinal epithelial cells (Int-407), normal amniotic cells (WISH), normal liver cells (Chang liver), laryngeal carcinoma (Hep-2), and oral cancer (KB). ). Hela cells are derived from a patient with cervical adenocarcinoma called Henrietta Lacks. Because of its extraordinary status, Hela cells are distributed around the world and are popular among laboratories. Therefore, until now, many scientists have not noticed the possibility of Hela cells being cross-contaminated with other cells. Hela cells are extremely strong and fast growing, and because they grow too fast, they quickly inhibit the growth of other cells.

Denial and complacency

For Gartler's discovery, some people and even those who should know more showed resistance and hostility, but one scientist, Walter Nelson-Rees, paid special attention to Gartler's words. Nelson-Rees contracted a cell bank affiliated with the National Cancer Institute. He and his colleagues developed a method for identifying cells using karyotyping. He published in a series of published articles: Sending to cells In the culture of the library, there is a large amount of cross-contamination in a unique cell culture. The work of Nelson-Rees shows that cross-contamination caused by Hela cells is extremely common, and even after many years, all cell lines may It is suspected to be a Hela cell unless it can be proven that it is not. Nelson-Rees not only raised the need to pay attention to cross-contamination in the scientific literature, he also informed the scientists affected by the problem by letter. Nelson-Rees's final contribution to cell identification was published in 2009 (not long after death).

When Nelson-Rees first published his findings, some scientists simply ignored or even denied his evidence and continued to publish academic papers containing the wrong information. As a result, Nelson-Rees was able to label papers or individuals using contaminated cell lines without any means. In that era (perhaps today), Nelson-Rees' behavior was considered unscientific and he was attacked by many colleagues. He advertised himself as a member of the Security Council. In 1981, NIH terminated his contract with him. Since then, the phenomenon of cell misidentification has intensified and the problem has gradually expanded. In the next 10 to 20 years, the cell bank distributed many misnamed cells.

It is difficult to assess how misleading and erroneous the study is caused by cross-contamination or cell misidentification. From 1969 to 2004, the use of misidentified cultures increased approximately 10-fold in the PubMed database, while papers published using cultured cells increased only 2-2.5 fold. By 2004, Hela cells were just the tip of the iceberg, and many other cell lines were prevalent in laboratories around the world under different “camouflage coats”.

A survey of workers working on cell culture showed that 32% of the 483 respondents used Hela cells, 9% used Hela contaminants without their knowledge, and only 33% of respondents used them. The cells were identified. 35% of people access cells in other laboratories, not large cell banks.

In the past more than 50 years, for the problem of cell misidentification, although some people will smug, and in some cases people's initial reaction is negative, there are still a small number of people who are committed to remedy this problem. The majority of the individual's efforts were written by the relevant staff to the editor to ask the reviewer to pay attention to the problem of cell misidentification, and the author was asked to provide evidence to prove that the cell line used in the study was neither cross-contaminated nor misidentified. . But these efforts were largely ignored after the termination of Nelson-Rees's contract, although the advent of DNA fingerprinting technology led to new and more regenerative methods that repeatedly revealed cell errors in the early 1990s. The degree of identification.

In 2004, Roland Nardone began his second reform campaign. He was supported by Joseph B. Perrone, who later became the vice chairman of the ATCC standards development organization. Joseph B. Perrone not only provided him with many ideas, but also provided Inspire the indignation of the reform movement. Together with other relevant scientists, Nardone proposes a comprehensive and coordinated initiative to simultaneously seek awareness of the nature and importance of the issue and the involvement of organizations and individuals affected by the issue, including NIH, Howard. Hughes Medical School.

Case and impact

Cross-contamination and misidentification have a long history, and there are many cases in which it is difficult to judge which one is the most important and the most expensive.

The classic case that has been described is Hela cell contamination (there are several cases of Hela cell contamination). Surprisingly, many cell lines contaminated with Hela cells are still used in some well-respected journals. One used them for the first time to find Hela cells for up to 40 years.

T24 is another fast-growing cell line that often contaminates many cell lines that are presumably derived from different bladder cancers. EVC304 was first thought to be a spontaneously transformed human normal endothelial cell line, but it was later found to be a T24 bladder cancer cell line. Surprisingly, the discussion that EVC304 is not an endothelial cell has little effect on its publication as an endothelial cell model.

It is speculated that human prostate cancer cell lines TSU-Pr1 and JCA-1 are also derived from the T24 bladder cancer cell line. These studies were published in the journal Cancer Research, but it did not prevent a subsequent study of TSU-Pr1 as a model of prostate cancer cell lines published in Cancer Research.

DNA fingerprinting revealed that the NCI/ADR-RES cell line was actually the ovarian tumor cell line OVCAR-8, rather than the breast cancer cell line. Approximately 300 published papers have used misidentified NCI/ADR-RES cell lines. NCI/ADR-RES is contained in NCI60 cells, and both STRs are identical.

A paper describing the misidentification of esophageal cell lines claims that experimental results based on these contaminated cells lead to continued clinical trials requiring recruitment of EAC (esophageal adenocarcinoma) patients, as well as access to more than 100 publications and at least three The NIH Oncology Institute acknowledges that it also involves 11 US patents.

50 years of suppression, why?

There are three groups that are responsible for the misidentification of cells: individual scientists, scientific journals, and money management departments. For most of the past 50 years, only scientists have protested this issue. However, it is difficult to get rid of the conclusion that many scientists deliberately use misidentified cell lines. In addition, authors are often reluctant to publish a statement based on the use of misidentified cells.

John Maddox, editor of Nature magazine, wrote a review in 1980 on the topic of cross-contamination, titled "Research is responsible for integrity." Although he had almost no insight into the problem, he made a suggestion: there is no reason to suspect that a few well-known cases are just the tip of the iceberg in any sense. In the same commentary, some scientists have been embarrassed like Nelson-Rees, because this article points out that if these civilized behaviors (ie, the issue of integrity in research) are claimed to be the destruction of members of the security team, then it will become a Disaster. The history of cross-contamination in cell lines suggests that integrity is not as applicable to the scientific community around the world as many scientists hope to believe.

The response of the editors of scientific journals related to this issue continues to be inspired. Hundreds of scientific journal articles describe examples of cell misidentification, but until now, scientific journals or money management departments have not taken action to eradicate this problem.

An editor of a very influential tissue culture journal was invited to consider cell identification as a necessary condition for publishing a paper, but he responded that it would be financial suicide. Editors of other journals also refused to consider this quality control program. They believe that the number of authors who are willing to contribute to their magazines will be greatly reduced once the barriers to publishing papers are introduced.

In the past two years, attitudes have begun to change, and some journals such as In Vitro Cellular and Developmental Biology, International Journal of Cancer, Cell Biochemistry, and Biophysics and the American Association for Cancer Research (AACR) journals require cell lines to be made before publication. Identification. Nature magazine pointed out: First, the IMF must ask cells to identify and provide the necessary funding. Once they do, Nature will ask for cell identification before the article is published. At the same time, the IMF continues to ignore this issue.

The IMF is the most powerful department to maintain scientific standards, but surprisingly, they refuse to consider dealing with or even recognizing cell misidentification. For example, the NIH announcement in 2007 ignored the failure of individual scientists and reviewers to resolve the problem of cell misidentification. A Nature editor pointed out that this announcement is only a forced acceptance of the status quo.

Some scientists personally tried to solve this problem, but they encountered a response from the fund department that did not help, they either tend to deny or weaken the problem. A recent statement from a senior scientist from Cancer Research UK stated that the problem of cell misidentification has risen every year. The fund part seems to have been threatened by this problem and has resisted the scientists trying to solve it, and the fund part has blamed and deprived scientists who are trying to find a solution.

Any major fund department supporting biomedical research in the United States or the United Kingdom should have eradicated the misidentification of cell lines in the past 10 years, and the need to eradicate this problem is less than that provided by the fund department published by science and society. Average funding for each project. However, these fund departments have repeatedly ignored or, in some cases, suppressed the debate on this issue and continue to fund research projects that use erroneous cells. There may be greater implications for the role of the fund department in distorting and deceiving science.

The problem of misidentification of cell lines should be intolerable for both journals and fund departments. There are signs that Nardone's “cracking” movement is constantly gaining influence, and the criteria for human cell line identification will be a clear evidence in the Nardone legacy.

The root cause of cell misidentification

Most cell lines are established in an academic setting. In an academic setting, tissue culture is often considered a technique that requires few skills, and the necessary equipment such as flow cabinets and incubators are used without restrictions. In these cases, it is not surprising to try to establish a new cell line that usually leads to cross-contamination. Of the 550 leukemia and lymphoma cell lines sent to the microbiology and cell culture collections in Germany, 59/395 (15%) were sent to the cell line by the initiators and 23/155 (15%) secondary sources it is wrong. It can be speculated that most of the secondary source cell lines have been cross-contaminated or misidentified by the initiator.

There are many reasons for misidentification of cell cultures, and there is a risk in every laboratory. Perhaps the most direct cause of cell misidentification is the mislabeling of cell culture vessels during routine operations. The causes of this problem are: the workload of the experimenter, lack of attention, and distraction during cell manipulation.

Cross-contamination of cultures and subsequent overgrowth of contaminating cells is another common cause of misidentification of cell lines. The probability of this occurring increases when the reagents are shared, the same tube is used repeatedly during the refeeding operation, and multiple cultures are simultaneously operated without adequately separating each cell type. When cross-contamination occurs, one cell type grows rapidly more than the other, and after 4-5 cell passages, a pure contaminating cell culture will form.

Intentional co-culture can result in cross-contamination or human cell line overgrowth when trophoblasts of other species (eg, mouse 3T3 cells) are used to propagate human stem cells or blast cells. Under normal circumstances, trophoblast cells are in a non-proliferating state, but as long as the growth inhibition program is incomplete, it will continue to multiply and gradually replace human cells. Somatic cell hybridization is not common but can occur. For example, the human mantle cell lymphoma cell line NCEB-1 carries seven chromosomes of mice.

Xenografts can also lead to cross-contamination and misidentification of cell lines, and regenerative cells from xenografts are replaced by host animal cells.

In general, the end result of cross-contamination will be a complete and rapid replacement of small amounts of adapted cells. The two cell lines cannot coexist in the same culture environment unless they are symbiotic, but this has not been reported to the best of our knowledge. A cell population can contain a stable mixed genome after many passages, the only known case being successive somatic cell hybridization.

Simple, economical quality control measures can prevent or at least reduce the occurrence of cellular misidentification. The lack of attention and the lack of quality control measures have led to the widespread misidentification of cells. The implementation of a large number of quality control measures and the appropriate adjustment of the corresponding documents are considered to be factors in the relatively low cell error identification report in the biopharmaceutical field.

Cross contamination detection

Many methods have been used to detect cross-contamination, including isozyme analysis, karyotyping, human leukocyte antigen (HLA) typing, immunophenotyping and DNA fingerprinting. These methods can identify a certain cell line, but the resolving power is different. However, the data obtained by these methods in different laboratories are not the same, so that no method can be used to establish a standard reference database.

Many laboratories use STR typing to identify human cell lines. STR typing is a method used for phylogenetic analysis, which is based on the simultaneous amplification of multiple polymorphic DNA fragments in a single tube. A STR locus is a repetitive DNA sequence consisting of a different number of repeating units. Each STR site can be amplified and the amplification products are labeled with different colors of fluorescence, making the amplification products readily distinguishable by fragment size and color. STR typing is fast, economical, easy to automate, provides repeatable data, and the data format is suitable for creating a standard reference database. Because of the rapid, well-identified cell line results, STR typing has the greatest value.

STR classification - potential and limitations

DNA repeats of 3-5 base repeats have been routinely used for phylogenetic identification, forensic identification, and identification of victims of a catastrophe 20 years ago. Subsequently, STR typing was used in cell identification. There are several advantages to using STR typing for cell identification.

Tumor cell lines contain many genetic alterations, so the criteria for analyzing tumor cell lines using STR typing must be different from normal tissue (see Supplementary Information Box 3). Tumor cells typically exhibit loss of heterozygosity (i.e., one allele is deleted so that it is difficult to distinguish from homozygotes), and in addition, due to DNA replication, tumor cells can carry multiple copies of the allele. Similarly, in tumor cell culture, a tumor cell line can either lose one copy of the allele or occasionally obtain a copy. Therefore, sublines of the same cell line may contain different STR types.

Comparing the same alleles, previously published data indicate that 75% of the consensus has been able to identify all known cross-contaminated cell lines, and two cell lines with more than 50% identity are not considered to be derived from different individuals. Thus, there is a 25% "buffer" between a single cell line and a cross-contaminated cell line, and any cell line should be considered suspicious as long as the level of identity is between 50% and 75%.

In analyzing human cell line genotypes, the main problems include heterozygous peak height imbalance (ie, the peak height or area of ​​an allele is much larger than another allele), multiple alleles at a locus The allele is lost (the fragment of interest is not amplified). Since tumor cell lines are aneuploid, the heterozygous peak height imbalance and the appearance of multiple alleles at one or more loci are typical.

The cost of genotyping is a major concern, but the cost is negligible relative to the entire work of the cell line. The cost of STR typing includes DNA extraction, PCR amplification of STR loci, capillary electrophoresis separation of amplified fragments, and data analysis. Increasing the number of STR loci, such as from 6 to 15, will achieve a very high resolution.

A major limitation of STR typing is that it cannot detect contaminating cells from other species. Even if human cells are covered by other species of cells that have been grown, there will be no DNA with human or higher primate-specific primers. Amplification. The use of species-specific primers in PCR can be used to detect contaminating cells from other species. If STR typing for other species has been established (currently limited to a few species of significant commercial value), STR typing can undoubtedly be used to identify contaminating cells.

For most of the established cell lines, donor tissue is no longer available, and those who are widely used are either originally retired or have died. In these cases, a hypothetical cell population based on the oldest stored in the cell bank appears to be imaginary. These typing will require a "temporary" label to indicate a lack of typing for the original donor tissue.

The available resources that can be used to compare STR typing are extremely limited until the database described below is available. The ATCC and DSMZ cell bank sites, as well as the CLIMA (Cellome Molecular Identification Integration Database), provide information on at least two series of STR typing data that have been published. Currently, one of the most useful resources is the list of misidentified cell lines collected by Amanda Capes-Davis and Ian Freshney, which is also available on the European Cell Culture Collection (ECACC) website. All researchers should check the list of cell lines they are using against this list.

Interactive database

It has been proposed to create a database designed to develop available STR typing data that can be used as data for human cell line identification. The interactive database is accessible to anyone, but only the database administrator can modify or supplement the data. This database not only provides DNA typing, but also allows the laboratory to compare the STR typing data of their cell lines with the cell line data in the database, which is convenient to ensure the reliability of the experimental data.

The universal standard is a prerequisite for composing a good database. The criteria for applying cell identification can establish an interactive database consisting of each single cell line with a defined STR classification, and provide a basis for STR typing and analysis. Condition. Members of the Standards Development Committee will work with NCBI to develop the conditions required for an interactive database maintained by NCBI. The database initially contained only 500 confirmed cell lines commonly used by researchers and kept in large cell banks.

Each cell line is determined for its STR typing before being submitted to the database. The most efficient database for comparing STR typing data consists of a common set of markers. However, not all data is collected from the same STR locus or from the same generation of sequencing instruments. The use of a primer-free combination for the same STR marker is a practice in forensic and human identification. The US Federal Bureau of Investigation's Joint DNA Indexing System (CODIS) uses a combination of 13 core STR loci to enter data. In order to maintain integration between CODIS data, laboratories must use CODIS-approved STR typing kits and instruments and operate in strict accordance with quality assurance standards. CODIS-approved kits have undergone extensive validation studies, including consistency studies designed to explain differences in STR typing when using different primer combinations. The same procedure would be necessary for cell STR typing.

future

From the increased data credibility, the time and expense required, and the effort spent on misidentified cell lines, cell line STR typing will have a major impact in scientific research. Accurate identification of cell lines plays a vital role in the development of cell-based medical products that avoid the risk of exposing human cells to misidentified cells. Although most of this misidentification can be avoided by following quality control measures, such as correct labeling, tracking programs when producing cell-based medical products, but when used to confirm a cellular product derived from the intended donor and it is intentionally When mixed with other donor cells, standard methods that clearly identify cells and tissues will provide security. This problem is very important for individualized medicine and the application of stem cell-based technologies, including induced pluripotent stem cells.

There is no single method that provides all the information to identify a human cell line. STR typing is the best representation of this period, so the STR typing standard will continue to improve as new information becomes available. An interactive, searchable database that is open to anyone will greatly reduce the use of misidentified cells. Fund departments and journals are also encouraged to adopt a “zero tolerance” policy for the use of misidentified cells and require authors to provide evidence to support their use of the correct cell line.

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