Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): National Research, Development and Innovation Office of Hungary (NKFIA; NVKP-16-1-2016-0017 National Heart Program and OTKA-FK 134751) MTA-SE System Pharmacology Research Group, Department of Pharmacology and Pharmacotherapy, Semmelweis University, H-1089 Budapest, Hungary Ischemic heart disease is the leading cause of death worldwide; therefore, the development of cardioprotective therapies is currently a main focus of research. Cultured cell lines and primary cell cultures play an integral role in cardiovascular research. However, there are many limitations when using these models, including variations in proliferation capacity, uncontrolled stress during cell isolation, poor reproducibility and low translational value. The aim of our study was to test the responsiveness of the most widely used cardiomyocyte platforms to simulated ischemia/reperfusion injury (SI/R) considering the effect of differentiation protocols. Human, rat and mouse cell lines as well as primary cell cultures were used for in vitro viability assay with or without widely used differentiation protocols. The cells were exposed to normoxic or simulated ischemia/reperfusion protocols: a simulated ischemic period of 6 hours was used for the primary and the differentiated cell cultures, while for the non-differentiated cells 16 hours of simulated ischemia followed by 2 hours of reperfusion was applied. The duration of simulated ischemia was determined from preliminary experiments. Viability of the cells was measured by calcein assay. In non-differentiated human AC16 and rat H9C2 cardiac cell lines, cell viability was significantly reduced (50%) by 16h SI / 2h R in contrast, the viability of the mouse HL-1 cell line was not reduced by 16 h SI. In primary rat neonatal cardiac myocytes 6h SI / 2h R caused significant cell death (25%). In primary human iPSC-derived cardiac myocytes 6h SI / 2h R did not result significant injury (10%) whereas 16h did (37%). Responsiveness of different cell types to SI/R ranged between 40-100%. We have shown that the responsiveness of primary cardiac cells and non-differentiated cardiac cell lines is significantly different to simulated ischemia / reperfusion. Differentiation protocols in cell lines markedly affects their response to simulated ischemia / reperfusion. Comparative analyses of different types of cardiomyocytes can provide a good basis for accurate design of in vitro cardioprotective test systems.

中文翻译：

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广泛使用的心肌细胞平台对模拟缺血/再灌注的反应性

资金致谢 资金来源类型：公共拨款——仅限国家预算。主要资金来源：匈牙利国家研究、发展和创新办公室 (NKFIA; NVKP-16-1-2016-0017 National Heart Program and OTKA-FK 134751) MTA-SE System Pharmacology Research Group, Department of Pharmacology and Pharmacotherapy , Semmelweis 大学, H-1089 布达佩斯, 匈牙利 缺血性心脏病是全世界死亡的主要原因；因此，心脏保护疗法的开发是目前研究的主要焦点。培养的细胞系和原代细胞培养在心血管研究中发挥着不可或缺的作用。然而，使用这些模型时存在许多限制，包括增殖能力的变化、细胞分离过程中不受控制的压力、重现性差和翻译价值低。我们研究的目的是测试最广泛使用的心肌细胞平台对模拟缺血/再灌注损伤 (SI/R) 的反应性，并考虑分化方案的影响。人、大鼠和小鼠细胞系以及原代细胞培养物用于体外活力测定，有或没有广泛使用的分化方案。将细胞暴露于常氧或模拟缺血/再灌注方案：对原代和分化细胞培养物使用 6 小时的模拟缺血期，而对于未分化细胞，模拟缺血 16 小时，然后再灌注 2 小时。应用。模拟缺血的持续时间由初步实验确定。通过钙黄绿素测定法测量细胞的活力。在未分化的人 AC16 和大鼠 H9C2 心肌细胞系中，16 小时 SI / 2 小时 R 细胞活力显着降低 (50%)，相比之下，小鼠 HL-1 细胞系的活力在 16 小时 SI 未降低。在原代大鼠新生心肌细胞中，6 小时 SI / 2 小时 R 导致显着的细胞死亡 (25%)。在原代人 iPSC 衍生的心肌细胞中，6 小时 SI / 2 小时 R 未导致显着损伤 (10%)，而 16 小时则 (37%)。不同细胞类型对 SI/R 的响应范围在 40-100% 之间。我们已经表明，原代心肌细胞和未分化心肌细胞系的反应性与模拟缺血/再灌注显着不同。细胞系中的分化方案显着影响它们对模拟缺血/再灌注的反应。不同类型心肌细胞的比较分析可以为准确设计体外心脏保护测试系统提供良好的基础。