Division of Translational Radiation Sciences, Department of Radiation Oncology, University of Maryland School of Medicine, Baltimore, Maryland 21201.
Radiat Res. 2022 Mar 1;197(3):209-217. doi: 10.1667/RADE-21-00148.1.
Cell line misidentification and contamination are major contributors to the reproducibility crisis in academic research. Authentication of cell lines provides assurances of the data generated; however, commercially available cells are often not subjected to rigorous identification testing. In this study, commercially available cell lines underwent testing to confirm cell identity and purity. The methods reported here outline the best practices for cell line authentication. Briefly, a commercially available primary rabbit aortic endothelial cell line was purchased for the intent of producing target proteins necessary for generating species-specific recombinant antibodies. These rabbit-specific antibodies would then be utilized for the development of in-house enzyme-linked immunosorbent assays (ELISA) to evaluate blood-based biomarkers of vascular injury after total-body irradiation. To authenticate the cell line, cell identity and purity were determined by single tandem repeat (STR) testing, flow cytometry, polymerase chain reaction (PCR), and cytochrome c oxidase subunit 1 (CO1) DNA Barcoding in-house and/or through commercial vendors. Fresh cells obtained from a New Zealand White rabbit (Charles River, Wilmington, DE) were used as a positive control. The results of STR and flow cytometry analyses indicated the cells were not contaminated with human or mouse cells, and that the cells were not of endothelial origin. PCR demonstrated that cells were also not of rabbit origin, which was further confirmed by a third-party vendor. An unopened vial of cells was submitted to another vendor for CO1 DNA Barcoding analysis, which identified the cells as being purely of bovine origin. Results revealed that despite purchase through a commercial vendor, the cell line marketed as primary rabbit aortic endothelial cells were of bovine origin. Purity analysis found cells were misidentified rather than contaminated. Further investigation to determine the cell type was not performed. The most cost-effective and efficient methodology for confirming cell line identity was found to be CO1 DNA Barcoding performed by a commercial vendor.
细胞系鉴定错误和污染是学术研究中重现性危机的主要原因。细胞系的鉴定可以确保数据的可靠性；然而，市售细胞通常没有经过严格的鉴定测试。在这项研究中，对市售细胞系进行了测试，以确认细胞的身份和纯度。本文报告的方法概述了细胞系鉴定的最佳实践。简而言之，购买了一种市售的原代兔主动脉内皮细胞系，用于生产产生种特异性重组抗体所需的靶蛋白。然后，这些兔特异性抗体将用于开发内部酶联免疫吸附测定（ELISA），以评估全身照射后血液中血管损伤的生物标志物。为了鉴定细胞系，通过单串联重复（STR）测试、流式细胞术、聚合酶链反应（PCR）和细胞色素 c 氧化酶亚单位 1（CO1）DNA 条形码（内部和/或通过商业供应商）确定细胞的身份和纯度。从新西兰白兔（Charles River，Wilmington，DE）获得的新鲜细胞用作阳性对照。STR 和流式细胞术分析的结果表明，细胞没有被人或鼠细胞污染，并且细胞不是内皮细胞来源。PCR 表明细胞也不是兔细胞来源，这一结果得到了第三方供应商的进一步证实。一个未开封的细胞瓶被提交给另一个供应商进行 CO1 DNA 条形码分析，该分析确定细胞纯粹是牛细胞来源。结果表明，尽管是通过商业供应商购买的，但被标记为原代兔主动脉内皮细胞的细胞系实际上是牛细胞来源。纯度分析发现细胞被错误鉴定而不是被污染。没有进一步调查细胞类型。最具成本效益和效率的确认细胞系身份的方法是通过商业供应商进行 CO1 DNA 条形码分析。
