Alam Perwez, Maliken Bryan D, Ivey Malina J, Jones Shannon M, Kanisicak Onur
Department of Pathology and Laboratory Medicine, College of Medicine, University of Cincinnati.
Department of Pathology and Laboratory Medicine, College of Medicine, University of Cincinnati;
J Vis Exp. 2020 Oct 10(164). doi: 10.3791/61073.
Ex vivo culture of the adult mammalian cardiomyocytes (CMs) presents the most relevant experimental system for the in vitro study of cardiac biology. Adult mammalian CMs are terminally differentiated cells with minimal proliferative capacity. The post-mitotic state of adult CMs not only restricts cardiomyocyte cell cycle progression but also limits the efficient culture of CMs. Moreover, the long-term culture of adult CMs is necessary for many studies, such as CM proliferation and analysis of gene expression. The mouse and the rat are the two most preferred laboratory animals to be used for cardiomyocyte isolation. While the long-term culture of rat CMs is possible, adult mouse CMs are susceptible to death and cannot be cultured more than five days under normal conditions. Therefore, there is a critical need to optimize the cell isolation and long-term culture protocol for adult murine CMs. With this modified protocol, it is possible to successfully isolate and culture both adult mouse and rat CMs for more than 20 days. Moreover, the siRNA transfection efficiency of isolated CM is significantly increased compared to previous reports. For adult mouse CM isolation, the Langendorff perfusion method is utilized with an optimal enzyme solution and sufficient time for complete extracellular matrix dissociation. In order to obtain pure ventricular CMs, both atria were dissected and discarded before proceeding with the disassociation and plating. Cells were dispersed on a laminin coated plate, which allowed for efficient and rapid attachment. CMs were allowed to settle for 4-6 h before siRNA transfection. Culture media was refreshed every 24 h for 20 days, and subsequently, CMs were fixed and stained for cardiac-specific markers such as Troponin and markers of cell cycle such as KI67.
成年哺乳动物心肌细胞（CMs）的体外培养为心脏生物学的体外研究提供了最相关的实验系统。成年哺乳动物CMs是终末分化细胞，增殖能力极小。成年CMs的有丝分裂后状态不仅限制了心肌细胞的细胞周期进程，还限制了CMs的高效培养。此外，成年CMs的长期培养对于许多研究来说是必要的，例如CM增殖和基因表达分析。小鼠和大鼠是用于心肌细胞分离的两种最常用实验动物。虽然大鼠CMs的长期培养是可行的，但成年小鼠CMs易死亡，在正常条件下培养不能超过五天。因此，迫切需要优化成年小鼠CMs的细胞分离和长期培养方案。采用这种改良方案，可以成功分离并培养成年小鼠和大鼠的CMs超过20天。此外，与先前的报道相比，分离的CM的siRNA转染效率显著提高。对于成年小鼠CM的分离，采用Langendorff灌注法，使用最佳酶溶液并给予足够时间以完全解离细胞外基质。为了获得纯心室CMs，在进行解离和铺板之前，将心房解剖并丢弃。将细胞分散在层粘连蛋白包被的培养板上，这有利于高效快速附着。在进行siRNA转染之前，让CMs沉降4 - 6小时。每24小时更换一次培养基，持续20天，随后，将CMs固定并染色，以检测心肌特异性标志物如肌钙蛋白和细胞周期标志物如KI67。
