The Saban Research Institute of Children's Hospital Los Angeles, Los Angeles, California, USA.
Nat Protoc. 2012 Jan 19;7(2):247-55. doi: 10.1038/nprot.2011.440.
We describe here a protocol for culturing epicardial cells from adult zebrafish hearts, which have a unique regenerative capacity after injury. Briefly, zebrafish hearts first undergo ventricular amputation or sham operation. Next, the hearts are excised and explanted onto fibrin gels prepared in advance in a multiwell tissue culture plate. The procedure allows the epicardial cells to outgrow from the ventricle onto a fibrin matrix in vitro. This protocol differs from those used in other organisms by using a fibrin gel to mimic blood clots that normally form after injury and that are essential for proper cell migration. The culture procedure can be accomplished within 5 h; epicardial cells can be obtained within 24-48 h and can be maintained in culture for 5-6 d. This protocol can be used to investigate the mechanisms underlying epicardial cell migration, proliferation and epithelial-to-mesenchymal transition during heart regeneration, homeostatic cardiac growth or other physiological processes.
我们在这里描述了一种从成年斑马鱼心脏培养心外膜细胞的方案，成年斑马鱼心脏在受伤后具有独特的再生能力。简要地说，斑马鱼心脏首先经历心室截断或假手术。接下来，将心脏取出并在事先准备好的多孔组织培养板中的纤维蛋白凝胶上进行体外培养。该过程允许心外膜细胞从心室向外生长到纤维蛋白基质上。该方案与其他生物体中使用的方案不同，它使用纤维蛋白凝胶来模拟受伤后通常形成的血栓，这对于细胞的正确迁移是必不可少的。培养过程可以在 5 小时内完成；心外膜细胞可在 24-48 小时内获得，并可在培养物中维持 5-6 天。该方案可用于研究心脏再生、心脏稳态生长或其他生理过程中心外膜细胞迁移、增殖和上皮-间充质转化的机制。
