Isolation, Transfection, and Long-Term Culture of Adult Mouse and Rat Cardiomyocytes.
Isolation, Transfection, and Long-Term Culture of Adult Mouse and Rat Cardiomyocytes.
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DOI：
10.3791/61073
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发表时间：
2020-10-10
期刊：
影响因子：
--
通讯作者：
Kanisicak O
中科院分区：
文献类型：
--
作者：
Alam P;Maliken BD;Ivey MJ;Jones SM;Kanisicak O
成年哺乳动物心肌细胞（CM）的离体培养是心脏生物学体外研究最相关的实验系统。成年哺乳动物CM是具有最小增殖能力的终末分化细胞。成年CM的有丝分裂后状态不仅限制了心肌细胞的细胞周期进程，而且限制了CM的有效培养。此外，成年CM的长期培养对于CM增殖和基因表达分析等许多研究是必要的。小鼠和大鼠是用于心肌细胞分离的两种最优选的实验室动物。虽然大鼠CM的长期培养是可能的，但成年小鼠CM容易死亡，并且在正常条件下不能培养超过五天。因此，迫切需要优化成年鼠CM的细胞分离和长期培养方案。通过这种改进的方案，可以成功地分离和培养成年小鼠和大鼠CM超过20天。此外，与以前的报道相比，分离的CM的siRNA转染效率显著增加。对于成年小鼠CM分离，使用Langendorff灌注方法，使用最佳酶溶液和足够的时间进行细胞外基质完全解离。为了获得纯心室CM，在进行分离和铺片之前解剖并丢弃两个心房。将细胞分散在层粘连蛋白包被的板上，这允许有效和快速的附着。在siRNA转染之前，使CM沉降4-6小时。每24小时更新培养基，持续20天，随后，将CM固定并针对心脏特异性标志物如肌钙蛋白和细胞周期标志物如KI 67染色。
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.
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影响因子：
3.7
作者：
Parente V;Balasso S;Pompilio G;Verduci L;Colombo GI;Milano G;Guerrini U;Squadroni L;Cotelli F;Pozzoli O;Capogrossi MC
通讯作者：
Capogrossi MC
影响因子：
20.1
作者：
Ackers-Johnson M;Li PY;Holmes AP;O'Brien SM;Pavlovic D;Foo RS
通讯作者：
Foo RS
影响因子：
4.6
作者：
Wang, Jinhu;Panakova, Daniela;Poss, Kenneth D.
通讯作者：
Poss, Kenneth D.
影响因子：
5.5
作者：
Dou, Ying;Arlock, Per;Arner, Anders
通讯作者：
Arner, Anders
影响因子：
1.2
作者：
Nippert, Franziska;Schreckenberg, Rolf;Schlueter, Klaus-Dieter
通讯作者：
Schlueter, Klaus-Dieter