Isolation and in vitro culture of primary cardiomyocytes from adult zebrafish hearts
Isolation and in vitro culture of primary cardiomyocytes from adult zebrafish hearts
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DOI：
10.1038/nprot.2013.041
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发表时间：
2013-04-01
期刊：
影响因子：
14.8
通讯作者：
Izpisua Belmonte, Juan Carlos
中科院分区：
文献类型：
--
作者：
Sander, Veronika;Sune, Guillermo;Izpisua Belmonte, Juan Carlos
该方案描述了如何从成年斑马鱼心脏中分离原代心肌细胞，并将其培养长达4周，从而将其用作体内实验的替代方法。胶原酶消化心室后，细胞暴露于增加钙浓度，以获得高纯度的心肌细胞。整个分离过程可在4-5小时内完成。我们建立的培养条件允许细胞保持其成熟的肌肉完整性和收缩特性。此外，培养的成年斑马鱼心肌细胞，与斑马鱼体内心脏再生类似，经历部分去分化，与哺乳动物的同类细胞相比，能够增殖。我们的方案能够研究接近天然心肌细胞的结构和功能特性，并允许应用在活体动物中不可行的体外技术和分析。
This protocol describes how to isolate primary cardiomyocytes from adult zebrafish hearts and culture them for up to 4 weeks, thereby using them as an alternative to in vivo experiments. After collagenase digestion of the ventricle, cells are exposed to increasing calcium concentrations in order to obtain high-purity cardiomyocytes. The whole isolation process can be accomplished in 4-5 h. The culture conditions we established allow the cells to preserve their mature sarcomeric integrity and contractile properties. Furthermore, adult zebrafish cardiomyocytes in culture, similarly to zebrafish in vivo heart regeneration, undergo partial dedifferentiation and, in contrast to their mammalian counterparts, are able to proliferate. Our protocol enables the study of structural and functional properties in close-to-native cardiomyocytes and allows the application of in vitro techniques and assays that are not feasible to perform in living animals.