A simple antegrade perfusion method for isolating viable single cardiomyocytes from neonatal to aged mice.
A simple antegrade perfusion method for isolating viable single cardiomyocytes from neonatal to aged mice.
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DOI：
10.14814/phy2.13688
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发表时间：
2018-05
影响因子：
2.5
通讯作者：
Matsuura H
中科院分区：
文献类型：
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作者：
Omatsu-Kanbe M;Yoshioka K;Fukunaga R;Sagawa H;Matsuura H
本研究的目的是建立一种简单且可重复的顺行灌注方法，用于分离单个存活的小鼠心脏细胞，并确定适合不同年龄小鼠的标准实用方案。通过使用输液泵从离体心脏的左心室心尖附近注射灌注液来进行顺行灌注，其中主动脉被夹紧。这可以通过冠状循环彻底灌注心肌。所有程序均在显微镜下在预热的加热垫上进行，从而可以监测注射和灌注过程。通过适当调整注射针的尺寸、酶溶液的成分和用量以及灌注流速，这种顺行灌注方法可以应用于新生至老年小鼠的心脏。我们检查了离体心室和心房肌细胞的形态特征和电生理特性，发现这些细胞与传统的基于 Langendorff 的逆行灌注方法获得的细胞大部分相同。与逆行灌注法类似，也从离心的上清液部分中同时分离出间质非肌细胞，例如心脏祖细胞。结果表明，无论小鼠年龄如何，通过本发明的顺行灌注方法都可以高质量地分离出单个心脏细胞。
The aim of this study was to establish a simple and reproducible antegrade perfusion method for isolating single viable mouse heart cells and to determine the standard practical protocols that are appropriate for mice of various ages. Antegrade perfusion was performed by injecting perfusate from near the apex of the left ventricle of the excised heart, the aorta of which was clamped, using an infusion pump. This could thoroughly perfuse the myocardium through the coronary circulation. All procedures were carried out on a prewarmed heater mat under a microscope, which allows for the processes of injection and perfusion to be monitored. With appropriate adjustment of the size of the injection needle, the composition and amount of enzyme solution and the perfusion flow rate, this antegrade perfusion method could be applied to the hearts of neonatal to aged mice. We examined the morphological characteristics and electrophysiological properties of the isolated ventricular and atrial myocytes and found that these cells were mostly identical to those obtained with the traditional Langendorff‐based retrograde perfusion method. Interstitial nonmyocytes, such as cardiac progenitor cells, were also isolated simultaneously from the supernatant fraction of the centrifugation, similar to the retrograde perfusion method. The results suggest that single heart cells can be well isolated with high degree of quality by the present antegrade perfusion method, regardless of the age of the mouse.