Cell Line Authentication Research Articles

Endometriotic cell culture contamination and authenticity: a source of bias in in vitro research?

STUDY QUESTIONAre the primary cell cultures and cell lines used in endometriosis research of sufficient quality?SUMMARY ANSWERPrimary cells used in endometriosis research lack purity and phenotypic characterisation, and cell lines are not genotypically authenticated.WHAT IS KNOWN ALREADYThe poor reproducibility of in vitro research and the lack of authenticity of the cell lines used represent reasons of concern in the field of reproductive biology and endometriosis research.STUDY DESIGN, SIZE, DURATIONIn the present study, past in vitro research in the field of endometriosis was systematically reviewed to determine whether the appropriate quality controls were considered. In addition, we explored the performance of Paired Box 2 (Pax2) as an endometrium specific marker in endometrial and endometriotic primary cell cultures; we also characterised the most diffused endometriosis cell lines with respect to important markers including the short tandem repeat (STR) profile.PARTICIPANTS/MATERIALS, SETTING, METHODSLiterature review part: almost 300 published protocols describing the isolation and creation of primary cell cultures from endometriosis were reviewed. Wet-lab part: primary cells isolated from 13 endometriosis patients were analysed by immunohistochemistry, immunofluorescence and FACS for the expression of Pax2. Cell lines Z11 and Z12, the most diffused endometriosis cell lines, were characterised with respect to the expression of Pax2, steroid hormone receptors and STR profile.MAIN RESULTS AND THE ROLE OF CHANCEFrom the literature review work, we underscored the lack of sufficient cell purity and phenotypic characterisation of primary cell cultures, which present high risk of contaminations from surrounding non-endometriotic tissues. Past work based on the use of cell lines was reviewed as well, and it emerged that cell line authentication was never performed.In an effort to address these weaknesses for future research, we present data on the performance of Pax2, a suitable marker to exclude ovarian (and other non-endometrial) cell contaminations from primary cell cultures; STR profiles of cell lines Z11 and Z12 were analysed and indicated that the cells were authentic. These profiles are now available for authentication purposes to researchers wishing to perform experiments with these cells.A quality control pipeline to assure sufficient quality of in vitro research in the field of reproductive biology and endometriosis is proposed. We encourage scientists, research institutes, journal reviewers, editors and funding bodies to raise awareness of the problem and adopt appropriate policies to solve it in the future.LARGE-SCALE DATASTR profiles of cell lines Z11 and Z12 are deposited at the Cellosaurus database—web.expasy.org.LIMITATIONS, REASONS FOR CAUTIONThere may be additional markers suitable to assess cell quality.WIDER IMPLICATIONS OF THE FINDINGSFuture in vitro research in endometriosis and the reliability of outcomes can be improved by using the recommendations presented in this study.STUDY FUNDING/COMPETING INTEREST(S)The study was partly financed by the ‘Stichting Fertility Foundation’ (The Netherlands). The authors declare no existing conflict of interest.TRIAL REGISTRATION NUMBERNon-applicable.

Approaching RNA-seq for Cell Line Identification

Cancer cell lines serve as invaluable model systems for cancer biology research and help in evaluating the efficacy of new therapeutic agents. However, cell line contamination and misidentification have become one of the most pressing problems affecting biomedical research. Available methods of cell line authentication suffer from limited access, time-consuming and often costly for many researchers, hence a new and cost-effective approach for cell line authentication is needed. In this regard, we developed a new method called CeL-ID for cell line authentication using genomic variants as a byproduct derived from RNA-seq data. CeL-ID was trained and tested on publicly available more than 900 RNA-seq dataset derived from the Cancer Cell Line Encyclopedia (CCLE) project; including most frequently used adult and pediatric cancer cell lines. We generated cell line specific variant profiles from RNA-seq data using our in-house pipeline followed by pair-wise variant profile comparison between cell lines using allele frequencies and depth of coverage values of the entire variant set. Comparative analysis of variant profiles revealed that they differ significantly from cell line to cell line whereas identical, synonymous and derivative cell lines share high variant identity and their allelic fractions are highly correlated, which is the basis of this cell line authentication protocol. Additionally, CeL-ID also includes a method to estimate the possible cross-contamination using a linear mixture model with any possible CCLE cells in case no perfect match was detected.

The Use of Short Tandem Repeat Analysis for Cell Line Authentication

Short tandem repeat analysis (STR) is a well-established international method of authentication and genetic stability testing of cell lines (CLs). Therefore, the development and introduction of this method into routine practice of cell banks and cell culture collections is a pressing concern. In addition, the expansion of the field of cell-line based biomedical cell products (BСPs) necessitates the implementation of STR as a tool of identification testing during quality control. The State Pharmacopoeia of the Russian Federation does not require mandatory use of STR for cell line identification, while other countries have been using this method for cell line quality control for about a decade. The use of identified CLs in medical practice will ensure the efficacy and safety of BCPs.The aim of the study was to assess the possibility of using STR analysis for authentication and genetic stability testing of CLs using U937, WISH, WIL2-S, NK-92, and Jurkat Clone E6-1 CLs as examples.Materials and me­thods: the following human CLs were used in the study: U937 (ECACC), WISH (ATCC), WIL2S (ATCC), NK-92 (ATCC), and Jurkat Clone E6-1 (ATCC). The CL allelic profiles were determined by STR using the COrDIS Plus kit (Gordiz, Russia). The electrophoretic separation was performed using a Genetic Analyzer 3500 Series instrument. The data provided on the websites of the European Collection of Authenticated Cell Cultures and American Type Culture Collection were used to compare the CL profiles.Results: the AuthentiFiler PCR Amplification Kit (Thermo Fisher Scientific, USA) and the GenePrint 10 System (Promega Corporation, USA) intended for CL authentication by STR were compared with the characteristics of the COrDIS plus kit (Gordiz, Russia). The results of the comparison demonstrated that the COrDIS plus kit includes all the loci found in the foreign kits, as well as the loci recommended by the International Cell Line Authentication Committee. The U-937, WIL2S, and NK-92 CLs demonstrated genetic identity with the reference profiles available on the websites of the international collections. The Jurkat Clone E6-1 CL was found to be genetically instable due to the loss of the amelogenin gene.Conclusions: it was demonstrated by the examples of U937, WISH, WIL2-S, NK-92, and Jurkat Clone E6-1 CLs that STR and the COrDIS plus kit could be used for authentication and genetic stability testing. The obtained results suggest the feasibility of using the COrDIS plus kit for the analysis of CLs used in BCPs, for BCP quality control, and biomedical research.

Immunophenotypic and Immunotypic Evalua-tions of Human Myeloma Cell Lines KMM-1, JJN3, LP1, L363, KMS-12BM and RPMI-8226 for Cell-Line Authentication

Immunophenotypic and Immunotypic Evalua-tions of Human Myeloma Cell Lines KMM-1, JJN3, LP1, L363, KMS-12BM and RPMI-8226 for Cell-Line Authentication

CLASTR: The Cellosaurus STR similarity search tool - A precious help for cell line authentication.

Despite an increased awareness of the problematic of cell line cross-contamination and misidentification, it remains nowadays a major source of erroneous experimental results in biomedical research. To prevent it, researchers are expected to frequently test the authenticity of the cell lines they are working on. STR profiling was selected as the international reference method to perform cell line authentication. While the experimental protocols and manipulations for generating a STR profile are well described, the available tools and workflows to analyze such data are lacking. The Cellosaurus knowledge resource aimed to improve the situation by compiling all the publicly available STR profiles from the literature and other databases. As a result, it grew to become the largest database in terms of human STR profiles, with 6,474 distinct cell lines having an associated STR profile (release July 31, 2019). Here we present CLASTR, the Cellosaurus STR similarity search tool enabling users to compare one or more STR profiles with those available in the Cellosaurus cell line knowledge resource. It aims to help researchers in the process of cell line authentication by providing numerous functionalities. The tool is publicly accessible on the SIB ExPASy server (https://web.expasy.org/cellosaurus-str-search) and its source code is available on GitHub under the GPL-3.0 license.

Intact-Cell MALDI-ToF Mass Spectrometry for the Authentication of Drug-Adapted Cancer Cell Lines.

The use of cell lines in research can be affected by cell line misidentification. Short tandem repeat (STR) analysis is an effective method, and the gold standard, for the identification of the genetic origin of a cell line, but methods that allow the discrimination between cell lines of the same genetic origin are lacking. Here, we use intact cell MALDI-ToF mass spectrometry analysis, routinely used for the identification of bacteria in clinical diagnostic procedures, for the authentication of a set of cell lines consisting of three parental neuroblastoma cell lines (IMR-5, IMR-32 and UKF-NB-3) and eleven drug-adapted sublines. Principal component analysis (PCA) of intact-cell MALDI-ToF mass spectrometry data revealed clear differences between most, but not all, of the investigated cell lines. Mass spectrometry whole-cell fingerprints enabled the separation of IMR-32 and its clonal subline IMR-5. Sublines that had been adapted to closely related drugs, for example, the cisplatin- and oxaliplatin-resistant UKF-NB-3 sublines and the vincristine- and vinblastine-adapted IMR-5 sublines, also displayed clearly distinctive patterns. In conclusion, intact whole-cell MALDI-ToF mass spectrometry has the potential to be further developed into an authentication method for mammalian cells of a common genetic origin.

Reply to ‘H-STS, L-STS and KRJ-I are not authentic GEPNET cell lines’

Reply to ‘H-STS, L-STS and KRJ-I are not authentic GEPNET cell lines’

H-STS, L-STS and KRJ-I are not authentic GEPNET cell lines

H-STS, L-STS and KRJ-I are not authentic GEPNET cell lines

Development of conventional and real time PCR assays for rapid species authentication of mammalian cell lines commonly used in veterinary diagnostic laboratories

Mammalian cell lines are valuable tools in biomedical fields, with applications ranging from disease diagnosis to the production of biological reagents and vaccines. Here we report the development of new conventional (cPCR) and real time PCR (qPCR) assays for species identification of several mammalian kidney cell lines originated from swine, green monkey, hamster and bovine tissues that are extensively used in veterinary diagnostic laboratories. The PCR primers and probes were selected from highly conserved mitochondrial genes and analyzed in silico by nucleotide BLAST in the National Center for Biotechnology Information (NCBI) website to ensure target specificity. The assays were highly species-specific and had no cross-reactivity against other tested cell lines originated from different mammalian species. Assay sensitivity (limit of detection; LOD) was determined using serial dilutions of cell line DNA as template. The estimated LODs were between 2.95 and 48 pg (picogram) DNA/assay for cPCR, and between 1.5 × 10−3 and 4.8 × 10−2 pg DNA/assay for qPCR. Multiplex qPCR assays were developed for simultaneous detection of up to three species in a single assay. The multiplex qPCR assays exhibited the same sensitivity as the corresponding singleplex assays with the exception of the green monkey species that demonstrated a 10–100 fold decline in the sensitivity. Contamination of swine cells was detected in one of the rabbit cell lines. The contamination was further confirmed by Sanger and Next-Generation sequencing.

Interlaboratory study to validate a STR profiling method for intraspecies identification of mouse cell lines.

The Consortium for Mouse Cell Line Authentication was formed to validate Short Tandem Repeat (STR) markers for intraspecies identification of mouse cell lines. The STR profiling method is a multiplex polymerase chain reaction (PCR) assay comprised of primers targeting 19 mouse STR markers and two human STR markers (for interspecies contamination screening). The goals of the Consortium were to perform an interlaboratory study to–(1) validate the mouse STR markers to uniquely identify mouse cell lines (intraspecies identification), (2) to provide a public database of mouse cell lines with the National Institute of Standards and Technology (NIST)-validated mouse STR profiles, and (3) to publish the results of the interlaboratory study. The interlaboratory study was an international effort that consisted of 12 participating laboratories representing institutions from academia, industry, biological resource centers, and government. The study was based on 50 of the most commonly used mouse cell lines obtained from the American Type Culture Collection (ATCC). Of the 50 mouse cell lines, 18 had unique STR profiles that were 100% concordant (match) among all Consortium laboratory members, and the remaining 32 cell lines had discordance that was resolved readily and led to improvement of the assay. The discordance was due to low signal and interpretation issues involving artifacts and genotyping errors. Although the total number of discordant STR profiles was relatively high in this study, the percent of labs agreeing on allele calls among the discordant samples was above 92%. The STR profiles, including electropherogram images, for NIST-validated mouse cell lines will be published on the NCBI BioSample Database (https://www.ncbi.nlm.nih.gov/biosample/). Overall, the interlaboratory study showed that the multiplex PCR method using 18 of the 19 mouse STR markers is capable of discriminating at the intraspecies level between mouse cell lines. Further studies are ongoing to refine the assay including (1) development of an allelic ladder for improving the accuracy of allele calling and (2) integration of stutter filters to identify true stutter.

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.

DkNET (NIDDK Information Network): Research tools that assist scientists in improving the rigor and reproducibility of their research

The use of problematic resources such as contaminated or misidentified cell lines and cross‐reactive antibodies lead to reproducibility problems. Research has shown that about twenty percent of published studies use contaminated cell lines [1]. Many factors, including the ability to easily retrieve alert information, result in the continued use of these resources wasting both time and money. Research Resource Identifiers (RRIDs) are unique identifiers for resources that assist finding, identifying, and tracking antibodies, cell lines, biological samples, organisms, and digital resources in the published literature [2]. RRIDs currently appear in over 8000 papers across 600 biomedical journals. Because authors have to supply RRIDs in their manuscripts ahead of publication, RRIDs provide a unique means for disseminating information about resource performance. For example, a search in the RRID portal (Nov 14, 2018) found 802 problematic cell lines (contaminated and/or misidentified cell lines) out of 101528 cell lines, as reported by the International Cell Line Authentication Committee (ICLAC). Analysis of cell lines in published papers using RRIDs vs those that didn't use RRIDs, showed the reported use of contaminated cell lines is significantly lower in papers with RRIDs than in the average paper [3], suggesting that making researchers aware of problematic resources may decrease their usage. To make it easier for researchers to find complete information, the NIDDK Information Network (dkNET.org) used RRIDs to develop several tools to help scientists enhance research rigor and reproducibility. dkNET introduced Resource Reports, a detailed overview of each resource identified by an RRID, including the characteristics of the resource, any warning and alert information, citation metrics from the biomedical literature and validation and rating information. Resource Reports let a researcher “follow” a resource, receiving up to date information on the usage of the resources they used (or created) in the literature. dkNET also offers a Rigor and Reproducibility tool that can generate an authentication report for grant applications in support of the NIH mandate on authentication of key biological resources. RRIDs and the associated tools and services make it easier for information on resource performance to be tracked and disseminated.Support or Funding InformationNIH NIDDK Grant U24DK097771This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Justification of Methodological Approaches to Identification Testing of Biomedical Cell Products

The manufacturer (developer) has to prepare a specification for each newly developed biomedical cell product (BCP) that has passed the stage of preclinical studies. The specification is included into the registration dossier when applying for marketing authorisation of a BCP. In accordance with the Order of the Ministry of Health of the Russian Federation No. 14n of 19 January 2017 «On approval of the specification format for a biomedical cell product» the specification should contain information about authenticity of the cell line used in the BCP, namely: morphological characteristics, expression of specific markers, expression of specific genes, expression of specific proteins, as well as markers of cell line stability. At present Russia has no practical experience in BCP quality evaluation. The aim of the study was to substantiate methodological approaches to authentication of cell lines used in BCPs as illustrated by quality evaluation of the DF-2 model cell line using test methods that allow for characterisation of the morphological, genetic, immunophenotypic, and cytogenetic profile of the cell line. Materials and methods: the study analysed the DF-2 cell line — human dermal fibroblasts (mesenchymal stem cells) obtained from the Institute of Cytology of the Russian Academy of Sciences (St. Petersburg). The following analytical test methods were used in the study: morphological analysis; flow cytometry for immunophenotyping of the DF-2 model cell line; short tandem repeats for creating an allelic profile of the model cell line; cytogenetic analysis — differential DAPI staining of metaphase chromosomes. Results: the paper summarises methodological approaches to identification testing of medicines containing living human cells (BCP analogues) currently used in international practice, and presents the results of authentication of the model cell line. Conclusions: methods used for BCP identification testing should ensure unambiguous authentication of the cell line according to its specification. The study performed helped to work out the procedure of authentication of a model cell line.

CeL-ID: cell line identification using RNA-seq data

BackgroundCell lines form the cornerstone of cell-based experimentation studies into understanding the underlying mechanisms of normal and disease biology including cancer. However, it is commonly acknowledged that contamination of cell lines is a prevalent problem affecting biomedical science and available methods for cell line authentication suffer from limited access as well as being too daunting and time-consuming for many researchers. Therefore, a new and cost effective approach for authentication and quality control of cell lines is needed.ResultsWe have developed a new RNA-seq based approach named CeL-ID for cell line authentication. CeL-ID uses RNA-seq data to identify variants and compare with variant profiles of other cell lines. RNA-seq data for 934 CCLE cell lines downloaded from NCI GDC were used to generate cell line specific variant profiles and pair-wise correlations were calculated using frequencies and depth of coverage values of all the variants. Comparative analysis of variant profiles revealed that variant profiles differ significantly from cell line to cell line whereas identical, synonymous and derivative cell lines share high variant identity and are highly correlated (ρ > 0.9). Our benchmarking studies revealed that CeL-ID method can identify a cell line with high accuracy and can be a valuable tool of cell line authentication in biomedical science. Finally, CeL-ID estimates the possible cross contamination using linear mixture model if no perfect match was detected.ConclusionsIn this study, we show the utility of an RNA-seq based approach for cell line authentication. Our comparative analysis of variant profiles derived from RNA-seq data revealed that variant profiles of each cell line are distinct and overall share low variant identity with other cell lines whereas identical or synonymous cell lines show significantly high variant identity and hence variant profiles can be used as a discriminatory/identifying feature in cell authentication model.

Incidences of problematic cell lines are lower in papers that use RRIDs to identify cell lines.

The use of misidentified and contaminated cell lines continues to be a problem in biomedical research. Research Resource Identifiers (RRIDs) should reduce the prevalence of misidentified and contaminated cell lines in the literature by alerting researchers to cell lines that are on the list of problematic cell lines, which is maintained by the International Cell Line Authentication Committee (ICLAC) and the Cellosaurus database. To test this assertion, we text-mined the methods sections of about two million papers in PubMed Central, identifying 305,161 unique cell-line names in 150,459 articles. We estimate that 8.6% of these cell lines were on the list of problematic cell lines, whereas only 3.3% of the cell lines in the 634 papers that included RRIDs were on the problematic list. This suggests that the use of RRIDs is associated with a lower reported use of problematic cell lines.

Isoenzyme and molecular approach for authenticating and monitoring of animal cell lines.

Authentication of cell lines is of paramount importance to validate the results from their use in biomedical research. Although isoenzyme polymorphism is the standard method, molecular methods based on mitochondrial DNA (mtDNA) have been developed to replace it. The aim of this study was the improvement of our isoenzyme electrophoretic analysis and the validation of one molecular technique targeted at mtDNA for the authentication of our animal cell lines. The combined method of cellular lysing through osmotic shock, followed by freezing-thawing in N2 to obtain isoenzyme extracts, and with 42 × 106 cells maintained the best efficiency. The superior electrophoretic conditions were PAGE run at 200 V. All cell lines had isoenzymatic mobility corresponding to their species to lactate dehydrogenase, malate-dehydrogenase, and glucose-6-phosphate dehydrogenase isoenzymes, and could be distinguished from each other. Two molecular techniques based on mtDNA were tested, one on the cytochrome b gene and other on cytochrome c oxidase I subunit gene. Due to difficulties in distinguishing all cell lines using only one these techniques, we merged the primers of two methods in such a way that there was a sufficient differentiation of all DNA fragments. The sequencing of these PCR products was also performed to validate these data.

Comprehensive characterization of RB1 mutant and MYCN amplified retinoblastoma cell lines

In retinoblastoma research tumor-derived cell lines remain an important model to investigate tumorigenesis and new therapy options, due to limited tumor material and lack of adequate animal models. A panel of 10 retinoblastoma cell lines was characterized with respect to mutation, methylation and expression of RB1 and MYCN. These established retinoblastoma cell lines represent the most frequent types of RB1 inactivation and together with the MYCN amplification status, three classes can be distinguished: RB1mut/MYCNnonA, RB1mut/MYCNA and RB1wt/MYCNA. MYCN amplification was identified in five cell lines, whereby two of them, RB522 and RB3823, harbor no aberration in RB1. Targeted sequencing of 160 genes often mutated in cancer identified only few variants in tumor-associated genes other than in RB1. None of these variants was recurrent. mRNA expression analyses of retinal markers, cell cycle regulators and members of the TP53 signaling pathway revealed a high variability between cell lines but no class-specific differences. The here presented thorough validation of retinoblastoma cell lines, including microsatellite analysis for cell line authentication, provides the basis for further in vitro studies on retinoblastoma.

Generating and working with Drosophila cell cultures: Current challenges and opportunities.

The use of Drosophila cell cultures has positively impacted both fundamental and biomedical research. The most widely used cell lines: Schneider, Kc, the CNS and imaginal disc lines continue to be the choice for many applications. Drosophila cell lines provide a homogenous source of cells suitable for biochemical experimentations, transcriptomics, functional genomics, and biomedical applications. They are amenable to RNA interference and serve as a platform for high-throughput screens to identify relevant candidate genes or drugs for any biological process. Currently, CRISPR-based functional genomics are also being developed for Drosophila cell lines. Even though many uniquely derived cell lines exist, cell genetic techniques such the transgenic UAS-GAL4-based RasV12 oncogene expression, CRISPR-Cas9 editing and recombination mediated cassette exchange are likely to drive the establishment of many more lines from specific tissues, cells, or genotypes. However, the pace of creating new lines is hindered by several factors inherent to working with Drosophila cell cultures: single cell cloning, optimal media formulations and culture conditions capable of supporting lines from novel tissue sources or genotypes. Moreover, even though many Drosophila cell lines are morphologically and transcriptionally distinct it may be necessary to implement a standard for Drosophila cell line authentication, ensuring the identity and purity of each cell line. Altogether, recent advances and a standardized authentication effort should improve the utility of Drosophila cell cultures as a relevant model for fundamental and biomedical research. This article is categorized under: Technologies > Analysis of Cell, Tissue, and Animal Phenotypes.

RF/6A Chorioretinal Cells Do Not Display Key Endothelial Phenotypes.

PurposeThe misuse of inauthentic cell lines is widely recognized as a major threat to the integrity of biomedical science. Whereas the majority of efforts to address this have focused on DNA profiling, we sought to anatomically, transcriptionally, and functionally authenticate the RF/6A chorioretinal cell line, which is widely used as an endothelial cell line to model retinal and choroidal angiogenesis.MethodsMultiple vials of RF/6A cells obtained from different commercial distributors were studied to validate their genetic, transcriptomic, anatomic, and functional fidelity to bona fide endothelial cells.ResultsTranscriptomic profiles of RF/6A cells obtained either de novo or from a public data repository did not correspond to endothelial gene expression signatures. Expression of established endothelial markers were very low or undetectable in RF/6A compared to primary human endothelial cells. Importantly, RF/6A cells also did not display functional characteristics of endothelial cells such as uptake of acetylated LDL, expression of E-selectin in response to TNF-α exposure, alignment in the direction of shear stress, and AKT and ERK1/2 phosphorylation following VEGFA stimulation.ConclusionsMultiple independent sources of RF/6A do not exhibit key endothelial cell phenotypes. Therefore, these cells appear unsuitable as surrogates for choroidal or retinal endothelial cells. Further, cell line authentication methods should extend beyond genomic profiling to include anatomic, transcriptional, and functional assessments.

Common Cell Lines Used to Study Bone Morphogenetic Proteins (BMPs).

Many research methods exist to elucidate the functions of BMPs during osteogenesis. This chapter briefly reviews common immortalized mesenchymal cell types used to measure the efficacy of osteogenic factors like BMP-2. Detailed information regarding media and culture conditions are provided. Parameters relevant to experimental reproducibility and cell line authentication are discussed.