组织工程与重建外科杂志 ›› 2023, Vol. 19 ›› Issue (2): 101-.

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利用 hiPSC-CPCs 结合纤维蛋白凝胶构建 3D 心肌微组织

  

  • 出版日期:2023-04-01 发布日期:2023-05-09

Construction of a  3D cardiac microtissues using hiPSC-CPCs combined with fibrin gel

  • Online:2023-04-01 Published:2023-05-09

摘要:

目的 利用人诱导多能干细胞来源心肌前体细胞(Human induced pluripotent stem cell-derived cardiac progenitorcells,hiPSCs-CPCs)结合纤维蛋白凝胶构建 3D 心肌微组织用于研究心脏发育及药物筛选。

方法 首先利用化学分化法获得 hiPSC-CPCs,并对其进行鉴定,然后将其混于纤维蛋白凝胶构建 3D 微组织,待 hiPSC-CPCs 分化成心肌细胞后获得心肌微组织 ;观察心肌微组织随培养时间延长的形态变化 ;分别于第 15 天和第 30 天对心肌微组织进行冰冻切片,使用免疫荧光对获得的心肌微组织进行心肌鉴定,并统计分析心肌微组织中心肌细胞的肌节长度、Z 带宽度和排列情况 ;使用钙成像技术检测第 15 天和第 30 天心肌微组织的钙信号变化 ;使用 MUSCLEMOTION ImageJ macro 插件分析第 15 天和第 30 天的心肌微组织的收缩情况,并利用该软件检测心肌微组织对药物的反应。

结果 使用化学分化法可高效分化获得 hiPSC-CPCs,将其与纤维蛋白凝胶结合可成功构建 3D 微组织,且该微组织培养至第 10 天可成功获得具有自发跳动能力的心肌微组织,继续培养至第 30 天,心肌微组织形态仍可维持,未出现溶解情况 ;免疫荧光结果显示,心肌微组织可高表达心肌标志物 cTnT 和 α-actinin,随着培养时间延长,肌节更趋于成熟状态 ;钙成像结果显示,可顺利检测到心肌微组织的钙信号变化,且随着培养时间延长,钙信号变化一致性明显提升 ;MUSCLEMOTION ImageJ macro 结果显示,随着培养时间延长,心肌微组织收缩频率降低、收缩幅度增强,且异丙肾上腺素能成功激动心肌微组织,且随培养时间的延长,心肌微组织对异丙肾上腺素的反应更加灵敏。结论 将 hiPSC-CPCs 混于纤维蛋白凝胶中可成功构建可持续培养的 3D 心肌微组织,其钙信号及收缩易于检测,且对药物反应敏感,有望成为研究心脏发育和药物筛选的有利工具。

关键词:

Abstract:

Objective To construct cardiac microtissues using hiPSC-CPCs combined with fibrin gel for heart development
research and drug screening. Methods Human induced pluripotent stem cells were differentiated into cardiac progenitor cells
(hiPSCs-CPCs) by chemical differentiation method, and they were identified before being mixed in fibrin gel to construct 3D
microtissues. Cardiac microtissues were obtained when hiPSC-CPCs were differentiated into hiPSC-CMs. The morphological
changes of cardiac microtissues were observed during the culture period. The cardiac microtissues on day 15th and 30th were fro
zen sectioned and identified by immunofluorescence. The sarcomere length, Z-line width and Z-line alignment of hiPSC-CMs in 
cardic microtissues were statistically analyzed. Calcium signal changes in cardiac microtissues on day 15th and 30th were detected
successfully by calcium imaging. MUSCLEMOTION ImageJ macro was used to analyze the contraction of cardiac microtissues on
day 15th and 30th , the responses of cardiac microtissues to drugs were also detected by this software. Results hiPSC-CPCs can
be efficiently differentiated using chemical differentiation method, and cardiac microtissues with spontaneous beating could be
obtained when the microtissues were cultured to day 10th . The cardiac microtissues could still maintain the morphology without
the occurance of dissolution after being cultured to day 30th . Immunofluorescence results showed that cardiac microtissues ex
pressed myocardial markers cTnT and α-actinin highly. And the statistical results showed that the sarcomeres’
structure tended
to be more mature with the prolongation of culture time. Calcium imaging showed that the calcium signals of cardiac microtissues
can be detected successfully, and the prolongation of culture time may improved the consistency of calcium signal. The results of
MUSCLEMOTION ImageJ macro showed that the contraction frequency of cardiac microtissues decreased, while the contraction
amplitude increased by prolonging the culture time. The cardiac microtissues could be stimulated by isoproterenol successfully.
And the longer the incubation time, the more obvious the reaction of cardiac microtissues to isoproterenol. Conclusion Fibrin
gel combined with hiPSC-CPCs can successfully construct cardiac microtissues which is sustainable, calcium signal and con
traction easily detectable, drug response sensitive, and promising to be a useful tool for studying cardiac development and drug
screening.

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