目的 建立一种基于心脏类器官模型的场电位测定方法，用于临床前药源性心脏毒性评价。方法 自组织法体外构建人诱导多能干细胞（hiPSC）的心脏类器官模型，显微镜下观察形态，培养至第18天（D18）免疫荧光法检测成纤维细胞标志物波形蛋白（VIM）、内皮细胞标志物（CD31）、心肌细胞标志物肌钙蛋白T（cTnT）的表达；选用钾通道阻滞剂E-4031作为阳性药评估方法的有效性，待心脏类器官状态稳定（D18≤培养时间≤ D30）、选取信号强度稳定且持续时间超过3 d的类器官分为对照组（不加药）、E-4031（0.5 μmol·L^-1）组，加药前1 h进行培养基更换，加药后使用微电极阵列技术对场电位进行检测，记录波形和电生理参数；应用建立的模型对美西律（3、10、30 μmol·L^-1）潜在的心律失常风险进行评估。结果 自D1起，hiPSC浓缩聚集呈球状，形成拟胚体，随着诱导分化时间延长，细胞球直径逐渐增加，球体边缘逐渐清晰；D8，球体产生自发性跳动； D18，心脏类器官状态稳定，细胞球体直径大小趋于稳定，直径为（1 499.03± 101.60） μm，形状规则，边缘光滑清晰，搏动频率规律，可达（33.39± 8.14）次·min^-1。VIM、CD31、cTnT在心脏类器官内高度表达，到达预期成熟特征。与对照组相比，加入0.5 μmol·L^-1的E-4031 1 h时，T波明显延长； 1 h起场电位持续时间（FPD）变化率显著升高（P＜0.05、0.01），校正场电位持续时间（FPDc）的变化率显著升高（P＜0.05）；搏动周期增加，3 h时达到较高幅度，尖峰振幅的变化率呈现出下降趋势，但差异均不显著。类器官暴露于3、10 μmol·L^-1美西律后，与对照组相比T波均无明显位移，当美西律浓度达30 μmol·L^-1时无场电位信号。与对照组相比，3 h时10 μmol·L^-1的美西律组FPDc变化率显著增加（P＜0.05）；搏动周期变化率在美西律浓度为10 μmol·L^-1时存在明显变化，3 h时达到较高幅度，存在显著性差异（P＜0.001）； 3 μmol·L^-1的美西律组尖峰振幅的变化率表现出明显的下降趋势，5 h时存在显著性降低（P＜0.05），当美西律浓度达到10 μmol·L^-1时，尖峰振幅的变化率愈加明显，1、3、5 h时呈现显著性下降（P＜0.05）。结论 建立的心脏类器官的场电位检测方法可以用于评估药物引起的心脏毒性，可在药物研发早期提供较为准确的数据支持。

Objective A field potential measurement method based on a heart organoid model was established for preclinical druginduced cardiotoxicity evaluation. Methods Human induced pluripotent stem cells (hiPSCs) were used to construct a heart organoid model in vitro by self-organization. The morphology was observed under a microscope. On day 18 (D18) of culture, the expression of fibroblast marker vimentin (VIM), endothelial cell marker CD31, and cardiomyocyte marker cardiac troponin T (cTnT) was detected by immunofluorescence. Potassium channel blocker E-4031 was used as a positive drug to evaluate the effectiveness of the method. When the heart organoids were in a stable state (D18 ≤ culture time ≤ D30), organoids with stable signal intensity and duration over three days were divided into a control group (without drug) and an E-4031 (0.5 μmol·L^-1) group. The culture medium was changed 1 h before drug administration. Field potentials were detected using microelectrode array technology after drug administration, and waveforms and electrophysiological parameters were recorded. The potential arrhythmogenic risk of mexiletine (3, 10, 30 μmol·L^-1) was evaluated using the established model. Results From D1, hiPSCs condensed and aggregated into spherical structures, forming embryoid bodies. As the induction differentiation time increased, the diameter of the cell spheres gradually increased, and the edges of the spheres became clearer. On D8, the spheres began to beat spontaneously. On D18, the heart organoids were in a stable state, the diameter of the cell spheres tended to be stable, with a diameter of (1 499.03 ± 101.60) μm, regular shape, smooth and clear edges, and regular beating frequency, reaching (33.39 ± 8.14) beats·min^-1. VIM, CD31, and cTnT were highly expressed in the heart organoids, reaching the expected mature characteristics. Compared with the control group, when 0.5 μmol·L^-1 E-4031 was added for 1 h, the T wave was significantly prolonged; the field potential duration (FPD) change rate significantly increased from 1 h (P < 0.05, 0.01), and the corrected field potential duration (FPDc) change rate significantly increased (P < 0.05); the beating period increased, reaching a higher amplitude at three hours, and the peak amplitude change rate showed a downward trend, but the differences were not significant. After exposure to 3 and 10 μmol·L^-1 mexiletine, there was no significant displacement of the T wave compared with the control group. When the mexiletine concentration reached 30 μmol·L^-1, there was no field potential signal. Compared with the control group, the FPDc change rate in the 10 μmol·L^-1 mexiletine group significantly increased at 3 h (P < 0.05); the beating period change rate showed a significant change at a mexiletine concentration of 10 μmol·L^-1, reaching a higher amplitude at 3 h, with a significant difference (P < 0.001); the peak amplitude change rate in the 3 μmol·L^-1 mexiletine group showed a significant downward trend, with a significant decrease at five hours (P < 0.05). When the mexiletine concentration reached 10 μmol·L^-1, the peak amplitude change rate became more obvious, showing significant decreases at 1, 3, and 5 h (P < 0.05). Conclusion The field potential detection method of heart organoids can be used to evaluate drug-induced cardiotoxicity and provide relatively accurate data support in the early stage of drug development.

R965

国家发改委支持先进制造业和现代服务业发展专项