Brand Nigel J, Lara-Pezzi Enrique, Rosenthal Nadia, Barton Paul J R
Imperial College London, National Heart & Lung Institute, Heart Science Centre, Harefield UB9 6JH, UK.
Methods Mol Biol. 2010;633:113-24. doi: 10.1007/978-1-59745-019-5_9.
We describe a method of isolating and maintaining primary cultures of mouse neonatal cardiac myocytes (NCM). This is derived from the well-established procedure for making NCM cultures from rat neonates by sequential digestion of rat ventricular myocardial pieces using a collagenase/pancreatin mixture. One-day-old mouse neonates are taken and the heart excised. The great vessels, atria, and top section of the ventricular chambers are cut away and the remaining ventricular myocardium is cut into small cubes (about 1-2 mm(3)). Heart pieces from at least 30 animals are then subjected to short (15-25 min) digestion in a shaking water bath in the presence of collagenase and pancreatin. Cell supernatants are taken and pooled together for a total of five digestion steps. The cells are then plated on gelatinized culture dishes and allowed to attach overnight. Myocyte cultures were inspected microscopically for up to 4 days, revealing that many myocytes beat throughout this period. This protocol may be of use for making primary cardiac myocyte cultures from transgenic mice and for investigating gene transcription and cell signalling.
我们描述了一种分离和维持小鼠新生心肌细胞（NCM）原代培养物的方法。这是从通过使用胶原酶/胰蛋白酶混合物对大鼠心室心肌片进行顺序消化来制备大鼠新生NCM培养物的成熟程序衍生而来的。取出生一天的小鼠新生仔并切除心脏。切除大血管、心房和心室腔的顶部部分，将剩余的心室心肌切成小方块（约1-2立方毫米）。然后将来自至少30只动物的心脏组织块在含有胶原酶和胰蛋白酶的摇床水浴中进行短时间（15-25分钟）消化。收集细胞上清液并合并在一起，共进行五个消化步骤。然后将细胞接种到明胶包被的培养皿上并使其附着过夜。在显微镜下检查心肌细胞培养物长达4天，发现许多心肌细胞在此期间都在跳动。该方案可能可用于从转基因小鼠制备原代心肌细胞培养物以及研究基因转录和细胞信号传导。
