Xu Xianghua, Colecraft Henry M
Department of Physiology and Cellular Biophysics, Columbia University, USA.
J Vis Exp. 2009 Jun 16(28):1308. doi: 10.3791/1308.
Cultured primary adult rodent heart cells are an important model system for cardiovascular research. Nevertheless, establishment of robust, viable cultured adult myocytes can be a technically challenging, rate-limiting step for many researchers. Here we described a protocol to obtain a high yield of adult rat heart myocytes that remain viable in culture for several days. The heart is isolated and perfused with collagenase and protease under low Ca2+ conditions to recover single myocytes. Ca2+-tolerant cells are obtained by stepwise increases in extracellular Ca2+ concentration in three subsequent wash steps. Cells are filtered, resuspended in culture medium, and plated on laminin coated slips. Cultured myocytes obtained using this protocol are viable for up to four days and are suitable for most experiments including electrophysiology, biochemistry, imaging and molecular biology.
原代培养的成年啮齿动物心脏细胞是心血管研究的重要模型系统。然而，对于许多研究人员来说，建立强健、有活力的成年心肌细胞培养体系在技术上可能具有挑战性，是一个限制实验进程的步骤。在此，我们描述了一种方法，可获得高产的成年大鼠心脏心肌细胞，这些细胞在培养中可存活数天。心脏被分离出来，并在低钙条件下用胶原酶和蛋白酶灌注，以获得单个心肌细胞。通过在随后的三个洗涤步骤中逐步提高细胞外钙浓度来获得耐钙细胞。细胞经过过滤，重悬于培养基中，然后接种到包被有层粘连蛋白的载玻片上。使用该方法获得的培养心肌细胞可存活长达四天，适用于包括电生理学、生物化学、成像和分子生物学在内的大多数实验。
