Isolation, Culture and Transduction of Adult Mouse Cardiomyocytes
Isolation, Culture and Transduction of Adult Mouse Cardiomyocytes
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DOI：
10.3791/54012
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发表时间：
2016-08-01
影响因子：
1.2
通讯作者：
Huang, Guo N.
中科院分区：
文献类型：
--
作者：
Judd, Justin;Lovas, Jonathan;Huang, Guo N.
培养的心肌细胞可用于使用与体内系统互补的技术来研究心肌细胞生物学。例如，体外培养的纯度和可获得性使得能够对生化分析、实时成像和电生理学进行精细控制。心肌细胞的长期培养提供了在短期培养中无法完成的额外实验方法。例如，去分化、细胞周期再进入和细胞分裂的体外研究迄今为止主要限于大鼠心肌细胞，其在长期培养中似乎更稳健。然而，丰富的转基因小鼠品系工具集和成熟的疾病模型的可用性使得小鼠系统对心脏研究具有吸引力。虽然有几个成年小鼠心肌细胞分离的报告，很少有研究表明他们的长期培养。本文介绍了一种逐步分离和长期培养成年小鼠心肌细胞的方法。首先，逆行Langendorff灌注用于用蛋白酶有效地消化心脏，然后进行重力沉降纯化。在分离后的一段时间的去分化后，细胞逐渐附着到培养物上，并且可以培养数周。腺病毒细胞裂解物用于有效地纯化分离的心肌细胞。这些方法为研究心脏生物学提供了一个简单而强大的模型系统。
Cultured cardiomyocytes can be used to study cardiomyocyte biology using techniques that are complementary to in vivo systems. For example, the purity and accessibility of in vitro culture enables fine control over biochemical analyses, live imaging, and electrophysiology. Long-term culture of cardiomyocytes offers access to additional experimental approaches that cannot be completed in short term cultures. For example, the in vitro investigation of dedifferentiation, cell cycle re-entry, and cell division has thus far largely been restricted to rat cardiomyocytes, which appear to be more robust in long-term culture. However, the availability of a rich toolset of transgenic mouse lines and well-developed disease models make mouse systems attractive for cardiac research. Although several reports exist of adult mouse cardiomyocyte isolation, few studies demonstrate their long-term culture. Presented here, is a step-by-step method for the isolation and long-term culture of adult mouse cardiomyocytes. First, retrograde Langendorff perfusion is used to efficiently digest the heart with proteases, followed by gravity sedimentation purification. After a period of dedifferentiation following isolation, the cells gradually attach to the culture and can be cultured for weeks. Adenovirus cell lysate is used to efficiently transduce the isolated cardiomyocytes. These methods provide a simple, yet powerful model system to study cardiac biology.