Explant culture of adult zebrafish hearts for epicardial regeneration studies.
Explant culture of adult zebrafish hearts for epicardial regeneration studies.
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DOI：
10.1038/nprot.2016.049
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发表时间：
2016-05
期刊：
影响因子：
14.8
通讯作者：
Poss KD
中科院分区：
文献类型：
--
作者：
Cao J;Poss KD
在这里，我们描述的方案培养成年斑马鱼心脏作为外植体，并研究体外再生心外膜组织。未受伤或受伤的成人心脏切除，清洗，并在培养箱中轻轻搅拌。心脏外植体的制备在2小时内完成，外植体可以在培养中维持30天或更长时间。利用立体荧光显微镜对外植体中的荧光转基因进行实时成像，监测心外膜细胞的动态行为。我们还描述了体外程序的遗传消融心外膜，细胞增殖试验，组织移植和头移植。执行这些方案需要基本的细胞培养和外科技术。与现有的在基质上培养分离的斑马鱼心外膜细胞的方法相反，本文描述的方法将心外膜细胞维持在完整的心脏表面，从而更好地实现体内细胞行为。我们的方案补充和扩展了心脏再生的体内研究。
Here we describe protocols to culture adult zebrafish hearts as explants, and to study regeneration of epicardial tissue ex vivo. Uninjured or injured adult hearts are excised, washed, and cultured in an incubator with gentle agitation. Preparation of heart explants is accomplished within 2 hours, and explants can be maintained in culture for 30 days or longer. Dynamic behaviors of epicardial cells are monitored through live imaging of fluorescent transgenes in the explant, using stereofluorescence microscopy. We also describe ex vivo procedures for genetic ablation of the epicardium, cell proliferation assays, tissue grafts, and bead grafts. Basic cell culture and surgical skills are required to carry out these protocols. As opposed to existing protocols for culturing isolated zebrafish epicardial cells on matrices, procedures described here maintain epicardial cells on an intact cardiac surface, better enabling in vivo cell behaviors. Our protocols complement and extend in vivo studies of heart regeneration.