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若干代H9C2大鼠心肌细胞对细胞毒性研究结果重复性的影响。

The effect of a number of H9C2 rat cardiomyocytes passage on repeatability of cytotoxicity study results.

作者信息

Witek Piotr, Korga Agnieszka, Burdan Franciszek, Ostrowska Marta, Nosowska Beata, Iwan Magdalena, Dudka Jarosław

机构信息

Independent Medical Biology Unit, Medical University of Lublin, Chodzki 6, 20-093, Lublin, Poland.

Department of Human Anatomy, Medical University of Lublin, Jaczewskiego 4, 20-090, Lublin, Poland.

出版信息

Cytotechnology. 2016 Dec;68(6):2407-2415. doi: 10.1007/s10616-016-9957-2. Epub 2016 Mar 5.

Abstract

The embryonic cardiomyocyte cell line H9C2 is commonly used in numerous in vitro studies, including cardiotoxicity analyses of new drugs. So far no results were published for studies on cell parameters variability during the cell line ageing process. For this reason the aim of the study was to evaluate the effect of a number of H9C2 rat embryonic cardiomyocytes passages on repeatability of study results for selected cytotoxicity parameters, with doxorubicin as a model toxic agent. The cultures were passaged twenty-five times. Cells from passage 1, 5, 10, 15, 20 and 25 were treated with doxorubicin for 24 h. Then drug cytotoxicity was evaluated with the MTT test and additionally the nuclear factor erythroid 2-related factor (Nrf2) gene expression was examined. The analysis of oxidative stress intensity and cell morphology was also assessed. The microscopic appearance of cells indicates that untreated cardiomyocytes morphology changes as well as sensitivity to toxic effects increases with the number of passages. Also an increase in oxidative stress in cells occurs with further passaging of cardiomyocytes. Statistical significance of differences in conducted tests results depended on doxorubicin concentration but in many cases the H9C2 line was found to be a reliable in vitro model only for the first five passages. For this reason it is important to take into consideration that further culturing of cardiomyocytes may not ensure repeatability of study results due to the culture ageing.

摘要

胚胎心肌细胞系H9C2常用于众多体外研究,包括新药的心脏毒性分析。到目前为止,尚未有关于该细胞系老化过程中细胞参数变异性研究的结果发表。因此,本研究的目的是以阿霉素作为模型毒性剂,评估H9C2大鼠胚胎心肌细胞传代次数对所选细胞毒性参数研究结果重复性的影响。培养物传代25次。用阿霉素处理第1、5、10、15、20和25代的细胞24小时。然后用MTT试验评估药物细胞毒性,并额外检测核因子红细胞2相关因子(Nrf2)基因表达。还评估了氧化应激强度和细胞形态分析。细胞的显微镜外观表明,未处理的心肌细胞形态变化以及对毒性作用的敏感性随传代次数增加而增加。随着心肌细胞进一步传代,细胞中的氧化应激也会增加。所进行测试结果差异的统计学显著性取决于阿霉素浓度,但在许多情况下,发现H9C2细胞系仅在前五代是可靠的体外模型。因此,重要的是要考虑到由于培养老化,心肌细胞进一步培养可能无法确保研究结果的重复性。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5a11/5101311/d22ca7bd4d88/10616_2016_9957_Fig1_HTML.jpg

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