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

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.

Three‐dimensional spherical gelatin bubble‐based scaffold improves the myotube formation of H9c2 myoblasts

“…In this study, we used the H9c2 cells, a rat cardiomyoblast cell line derived from the embryonic rat ventricle, to demonstrate the influences of dimensionality to the differentiation of muscle cells. H9c2 cardiomyoblasts showed similar hypertrophic responses to primary neonatal cardiomyocyte cells, which are used to study the drug metabolizing enzymes in the heart (Watkins, Borthwick, & Arthur, 2011;Witek et al, 2016;Zordoky & El-Kadi, 2007). Some groups also used H9c2 cells to study cardiac muscle tissue engineering (Wang et al, 2018).…”

Toxic effect of high glucose on cardiomyocytes, H9c2 cells: Induction of oxidative stress and ameliorative effect of trolox

“…In our study, alterations in (Ca 2+ ) i followed the same pattern as seen in ROS generation under the same experimental conditions, indicating a probable crosstalk between the two messengers ( Our study demonstrates that H9c2 cells are more relevant for study on energy metabolism and mitochondrial function as of intact heart. [45] Nevertheless, uncertainty exists in determining the maximum number of passages required for in vitro studies. [42] Furthermore, human AC16 cardiac cells exposed to different concentrations of high glucose at 24 hours have shown decreased viability [43] similar to H9c2 cells in the present study.…”

“…Although H9c2 cells and primary neonatal cardiomyocytes show similar responses in viro, [44] the cell line aging process plays an important role in cardiotoxicity studies in H9c2 cells. [45] Nevertheless, uncertainty exists in determining the maximum number of passages required for in vitro studies.…”

Tetramethylpyrazine reverses high-glucose induced hypoxic effects by negatively regulating HIF-1α induced BNIP3 expression to ameliorate H9c2 cardiomyoblast apoptosis

“…H9c2 cell lines show a identical hypertrophic responses as those observed in primary neonatal cardiomyocytes when exposed to hypertrophic factors like angiotensin-II, endothelin-1 [19]. The cell line is also used for cardiotoxicity analyses and to understand stress associated mechanisms and myocyte damage including apoptosis and necrosis [20]. Moreover, H9c2 cells are well known to mimic primary cardiomyocytes in their responses to hypoxia and in addition, they are more similar to primary cardiomyocytes in their energy metabolism patterns.…”