Authentication Of Human Cell Lines Research Articles

SAT-414 Cytogenetic, Genomic, and Functional Characterization of Pituitary Gonadotrope Cell Lines

Cell lines have been widely used as models to characterize important intracellular and molecular mechanisms. Funding agencies and publishers – including the National Institutes of Health and Nature – have required researchers to authenticate human cell lines to establish their identity. Nevertheless, authentication of human cell lines has been poorly reported, let alone that of nonhuman species [1]. The use of different clones by diverse laboratories may lead to irreproducible results. Moreover, one should not assume that cell lines with the same name actually display identical characteristics, as differences in storage and culturing conditions may promote genomic variability and clonal divergence. In the reproductive field, the LβT2 [2] and αT3-1 [3] cell lines represent invaluable models for studying the regulation of pituitary gonadotropes by gonadotropin releasing hormone (GnRH) and other hormones. Both lines were produced by targeted oncogenesis in transgenic mice, with the LβT2 cells representing developmentally more mature gonadotropes than the αT3-1 cells. To establish a reference for each gonadotrope line, we requested early aliquots from the laboratory that generated them. Authentication by 27-dinucleotide and 9-tetranucleotide short tandem repeat (STR) genotyping was consistent with a mixed C57BL/6J x BALB/cJ genetic background. Spectral karyotyping revealed a variable number of chromosomes amongst cells, a result confirmed by shallow single-cell DNA sequencing in LβT2 cells. Interestingly, STR genotyping of several LβT2 batches obtained regularly throughout the past 20 years in our laboratory as well as in other laboratories identified a strain (#2) with a profile distinct from our reference line (#1). The two lines differed morphologically, with strain #2 exhibiting larger cells and extensions, whereas strain #1 displayed smaller uniform rounded cells. Moreover, the two lines showed variations in basal and GnRH-induced expression of early response and gonadotropin subunit genes. In line #1, under continuous GnRH stimulation, Egr1, Fos, and Lhb transcript levels were higher, whereas time course studies revealed lower Fos expression and no Fshb induction. Under a low-frequency regimen of pulsatile GnRH favoring Fshb induction, line #1 showed higher Egr1 transcript levels, but an absence of Fshb induction. Our results suggests that LβT2 strains exist that differ genetically and in their expression profiles. Our study demonstrates the importance of characterizing gonadotrope cell lines to improve data reproducibility, design and interpretation of gene expression experiments while continuing to use these cell lines that are important models for the study of gonadotrope physiology. [1] Almeida JL et al. PLoS Biol., 2016, 14(6):e1002476. [2] Thomas P et al. Endocrinology, 1996, 137(7): 2979-89. [3] Windle JJ et al. Mol. Endocrinology, 1990, 4(4): 597-603.

SNP array profiling of mouse cell lines identifies their strains of origin and reveals cross-contamination and widespread aneuploidy.

BackgroundThe crisis of Misidentified and contaminated cell lines have plagued the biological research community for decades. Some repositories and journals have heeded calls for mandatory authentication of human cell lines, yet misidentification of mouse cell lines has received little publicity despite their importance in sponsored research. Short tandem repeat (STR) profiling is the standard authentication method, but it may fail to distinguish cell lines derived from the same inbred strain of mice. Additionally, STR profiling does not reveal karyotypic changes that occur in some high-passage lines and may have functional consequences. Single nucleotide polymorphism (SNP) profiling has been suggested as a more accurate and versatile alternative to STR profiling; however, a high-throughput method for SNP-based authentication of mouse cell lines has not been described.ResultsWe have developed computational methods (Cell Line Authentication by SNP Profiling, CLASP) for cell line authentication and copy number analysis based on a cost-efficient SNP array, and we provide a reference database of commonly used mouse strains and cell lines. We show that CLASP readily discriminates among cell lines of diverse taxonomic origins, including multiple cell lines derived from a single inbred strain, intercross or wild caught mouse. CLASP is also capable of detecting contaminants present at concentrations as low as 5%. Of the 99 cell lines we tested, 15 exhibited substantial divergence from the reported genetic background. In all cases, we were able to distinguish whether the authentication failure was due to misidentification (one cell line, Ba/F3), the presence of multiple strain backgrounds (five cell lines), contamination by other cells and/or the presence of aneuploid chromosomes (nine cell lines).ConclusionsMisidentification and contamination of mouse cell lines is potentially as widespread as it is in human cell culture. This may have substantial implications for studies that are dependent on the expected background of their cell cultures. Laboratories can mitigate these risks by regular authentication of their cell cultures. Our results demonstrate that SNP array profiling is an effective method to combat cell line misidentification.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-847) contains supplementary material, which is available to authorized users.

Cell Line Contamination and Misidentification

Geneva Foundation for Medical Education and Research, Versoix, SwitzerlandDear Editors:RE: Cell line contamination and misidentificationIn order to further knowledge in the area of reproductivebiology, endocrinology, and immunology in a meaningfulmanner, researchers should be aware of cell line contaminationand misidentification, which is commonplace [1, 2]. Last yearKorch et al. [3] reported that this is the case for a number ofwidely used endometrial and ovarian cell lines.I, therefore, wish to highlight two issues:1. The American Type Culture Collection (ATCC) Recom-mendations. The ATCC has recently published a Standard forhuman cell line authentication, recommending short tandemrepeat (STR) profiling for authentication of human cell lines toaddress this problem [4].2. The paucity of endometrial epithelial cell lines available.Korch et al. identified EEC1 cells as being Ishikawa cells,MCF-7 cells or a mixture of both; hTERT EEC cells,previously thought to be normal endometrial epithelial cells,as being MCF-7 breast cancer epithelial cells; and HES cells asHeLa cervical cancer cells.It is advisable to alert researchers in training to theimportance of not just authenticating new cell lines uponreceipt but taking all necessary technical precautions in order toprevent cell line contamination as well as evaluating cell linesfor phenotypic or functional traits, which can be altered withdifferentiation and increasing passages [5].Yours sincerely,Geraldine O. Canny Ph.D.REFERENCES

Match criteria for human cell line authentication: Where do we draw the line?

Continuous human cell lines have been used extensively as models for biomedical research. In working with these cell lines, researchers are often unaware of the risk of cross-contamination and other causes of misidentification. To reduce this risk, there is a pressing need to authenticate cell lines, comparing the sample handled in the laboratory to a previously tested sample. The American Type Culture Collection Standards Development Organization Workgroup ASN-0002 has developed a Standard for human cell line authentication, recommending short tandem repeat (STR) profiling for authentication of human cell lines. However, there are known limitations to the technique when applied to cultured samples, including possible genetic drift with passage. In our study, a dataset of 2,279 STR profiles from four cell banks was used to assess the effectiveness of the match criteria recommended within the Standard. Of these 2,279 STR profiles, 1,157 were grouped into sets of related cell lines-duplicate holdings, legitimately related samples or misidentified cell lines. Eight core STR loci plus amelogenin were used to unequivocally authenticate 98% of these related sets. Two simple match algorithms each clearly discriminated between related and unrelated samples, with separation between related samples at ≥80% match and unrelated samples at <50% match. A small degree of overlap was noted at 50-79% match, mostly from cell lines known to display variable STR profiles. These match criteria are recommended as a simple and effective way to interpret results from STR profiling of human cell lines.

High-throughput SNP-based authentication of human cell lines.

Use of false cell lines remains a major problem in biological research. Short tandem repeat (STR) profiling represents the gold standard technique for cell line authentication. However, mismatch repair (MMR)-deficient cell lines are characterized by microsatellite instability, which could force allelic drifts in combination with a selective outgrowth of otherwise persisting side lines, and, thus, are likely to be misclassified by STR profiling. On the basis of the high-throughput Luminex platform, we developed a 24-plex single nucleotide polymorphism profiling assay, called multiplex cell authentication (MCA), for determining authentication of human cell lines. MCA was evaluated by analyzing a collection of 436 human cell lines from the German Collection of Microorganisms and Cell Cultures, previously characterized by eight-loci STR profiling. Both assays showed a very high degree of concordance and similar average matching probabilities (~1 × 10(-8) for STR profiling and ~1 × 10(-9) for MCA). MCA enabled the detection of less than 3% of contaminating human cells. By analyzing MMR-deficient cell lines, evidence was obtained for a higher robustness of the MCA compared to STR profiling. In conclusion, MCA could complement routine cell line authentication and replace the standard authentication STR technique in case of MSI cell lines.

Abstract 4256: Use of short tandem repeat profiling for authentication of human cell lines

Abstract Human cell lines are widely used in biological and biomedical research, and accurate data interpretation depends on unambiguous identity of a cell line relative to its original source. Contamination and misidentification of human cell cultures has been identified as a long-standing issue within the scientific community, and steps are now being taken to address the problem. Short-tandem-repeat (STR) DNA profiling provides an accurate, reliable, and standardized method for authentication of human identity. Currently, the ATCC is considering proposed standards for cell line authentication utilizing human STR typing, and many journals (including all AACR published journals) have adopted the NIH-recommended policy to mandate cell line authentication as a requirement for publication. Here we demonstrate the validation and use of two different STR systems to authenticate human cell lines as well as detect contamination by mouse feeder cells and other human cell lines. Threshold levels for detection of complete profiles as well as contamination are compared between the systems, and typical typing issues specific to cell lines are discussed. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4256. doi:1538-7445.AM2012-4256

Human, mouse or rat? Species authentication of glioma-derived cell cultures

Cell culture and the use of cell lines are often fundamental requirements in basic scientific research. It is of the utmost importance for researchers to ensure that the cell lines in use have a well defined origin and are routinely re-analysed to highlight possible areas of contamination. In this preliminary study species specific primers were designed to easily distinguish between human, mouse and rat DNA with standard agarose gel electrophoresis. Inter-species contamination is often the most common form of contamination experienced, with the most common of cell lines in use being of human, mouse and rat derivation. A PCR-based assay was therefore developed to ensure an accurate, quick and cost effective means of determining any cell line contamination which could be easily executed on a routine basis. Furthermore, this simple PCR is able to identify the species in the inter-species mixture of DNA and therefore provides a valuable tool for the authentication of human cell lines.

Cell line misidentification: the beginning of the end

Cell lines are used extensively in research and drug development as models of normal and cancer tissues. However, a substantial proportion of cell lines is mislabelled or replaced by cells derived from a different individual, tissue or species. The scientific community has failed to tackle this problem and consequently thousands of misleading and potentially erroneous papers have been published using cell lines that are incorrectly identified. Recent efforts to develop a standard for the authentication of human cell lines using short tandem repeat profiling is an important step to eradicate this problem.

Cell Line Data Base: structure and recent improvements towards molecular authentication of human cell lines

The Cell Line Data Base (CLDB) is a well-known reference information source on human and animal cell lines including information on more than 6000 cell lines. Main biological features are coded according to controlled vocabularies derived from international lists and taxonomies. HyperCLDB (http://bioinformatics.istge.it/hypercldb/) is a hypertext version of CLDB that improves data accessibility by also allowing information retrieval through web spiders. Access to HyperCLDB is provided through indexes of biological characteristics and navigation in the hypertext is granted by many internal links. HyperCLDB also includes links to external resources. Recently, an interest was raised for a reference nomenclature for cell lines and CLDB was seen as an authoritative system. Furthermore, to overcome the cell line misidentification problem, molecular authentication methods, such as fingerprinting, single-locus short tandem repeat (STR) profile and single nucleotide polymorphisms validation, were proposed. Since this data is distributed, a reference portal on authentication of human cell lines is needed. We present here the architecture and contents of CLDB, its recent enhancements and perspectives. We also present a new related database, the Cell Line Integrated Molecular Authentication (CLIMA) database (http://bioinformatics.istge.it/clima/), that allows to link authentication data to actual cell lines.

Antibody variable-region sequencing as a method for hybridoma cell-line authentication

Cross-contamination and misidentification of various cell lines is a widespread problem that can lead to spurious scientific conclusions. DNA fingerprinting is a powerful identification technique, which can be effectively used for the authentication of human cell lines. In contrast to human cancer cell lines, little attention has so far been given to establishing authentication practices for hybridoma cell lines. Since the majority of hybridomas stem from inbred animals, they have high genetic uniformity, which reduces the applicability of DNA fingerprinting. In the present study, we propose antibody variable-region sequencing as a method of choice for hybridoma cell-line authentication. This method focuses on the most diverse characteristic of hybridoma cell lines and thereby achieves a very high discriminatory power. The sequencing of light-chain variable regions has proven to be especially suitable for routine use because of its high success rate. Two other possible authentication methods, random amplified polymorphic DNA analysis and two-dimensional gel electrophoresis, were also examined. Compared to these and other methods that can be used for discrimination between hybridoma cell lines, variable-region sequencing has many advantages, most notably those of a very high discriminatory power, insensitivity to changes in experimental conditions, simple data analysis, and accessibility to most laboratories.

Profiling and authentication of human cell lines using short tandem repeat (STR) loci: Report from the National Cell Bank of Iran

Assurance of cell line homogeneity and capability of cell contamination detection are among the most essential steps of cell based research. Due to high discriminatory efficiency, low cost and reliability, analysis of short tandem repeats (STR) has been introduced as a method of choice for human cell line authentication. In the present study 13 Combined DNA Index System (CODIS) based STRs along with the gender determination (Amelogenin) gene were utilized to establish a reproducible approach for the authentication of 100 human cell lines deposited in the National Cell Bank of Iran (NCBI), using the polymerase chain reaction (PCR) method. PCR products were subsequently analyzed by polyacrylamide gel electrophoresis (PAGE) and visualized by silver staining followed by gel documentation and software analysis. STR profiles obtained were compared with those of the American Type Culture Collection (ATCC) and the Japanese Collection of Research Bioresource (JCRB) as STR references. We detected 18.8% cross contamination among the NCBI human cell lines. To our knowledge, this is the first report of authentication of human cell lines using the 13 CODIS core STRs combined with Amelogenin.

Essential role for gene profiling analysis in the authentication of human cell lines

Cross-contamination between cultured cell lines can result in the generation of erroneous scientific data. Hence, it is very important to eliminate cell lines that are of an origin different from that being claimed. Inter-species contamination can be detected by various established methods, such as karyotype and isozyme analyses. However, it has been impossible to detect intraspecies cross-contamination prior to the development of technology to detect differences between cell lines at the molecular level. Recently, profiling of short tandem repeat (STR) polymorphisms has been established as a method for the analyses of gene polymorphism. Gene profiling by STR polymorphism (STR profiling) is a simple and reliable method to identify individual cell lines. Each human cell line currently provided by the Cell Engineering Division of the RIKEN BioResource Center was analyzed by STR profiling to authenticate its identity. We found that more than 10 human cell lines out of approximately 400 were in fact identical to a different cell line deposited in the collection, and therefore had been misidentified. We conclude that STR profiling is a useful and powerful method for eliminating cell lines that have been misidentified by cross-contamination or by other causes. Hence, STR profiling of human cell lines used in published research will likely be a prerequisite for publication in the future, so that the problem of misidentification of cell lines can be eliminated.