A Simplified, Langendorff-Free Method for Concomitant Isolation of Viable Cardiac Myocytes and Nonmyocytes From the Adult Mouse Heart.
A Simplified, Langendorff-Free Method for Concomitant Isolation of Viable Cardiac Myocytes and Nonmyocytes From the Adult Mouse Heart.
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DOI：
10.1161/circresaha.116.309202
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发表时间：
2016-09-30
影响因子：
20.1
通讯作者：
Foo RS
中科院分区：
文献类型：
--
作者：
Ackers-Johnson M;Li PY;Holmes AP;O'Brien SM;Pavlovic D;Foo RS
心血管疾病是一种全球性流行病。小鼠基因组学、表观基因组学和转基因学的出现和最新进展为强大的研究途径提供了更大的潜力。然而，进展往往受到与成年小鼠心脏活肌细胞分离相关的独特复杂性的限制。目前的方案依赖于使用专门的Langendorff装置的逆行主动脉灌注，这对研究人员造成了相当大的后勤和技术障碍，并需要大量的培训投资。确定并优化一种方便的替代方法，仅使用普通手术和实验室设备即可稳健分离和培养成年小鼠心肌细胞。分离心肌细胞，其产率与已发表的基于Langendorff的方法相当，使用LV离体直接针灌注，无需肝素注射。分离的肌细胞可以在无抗生素的情况下培养，保留有组织的收缩和线粒体形态、转录特征、钙处理、对缺氧的反应、神经激素刺激和电起搏，并且适合于膜片钳和腺病毒基因转移技术。此外，该方法允许同时分离、分离和共培养肌细胞和非肌细胞心脏群体。我们提出了一种新的，简化的方法，证明伴随分离可行的心肌细胞和非心肌细胞从同一成年小鼠心脏。我们预计，这种新方法将扩大和加速心脏生物学领域的创新研究。
Cardiovascular disease represents a global pandemic. The advent of and recent advances in mouse genomics, epigenomics, and transgenics offer ever-greater potential for powerful avenues of research. However, progress is often constrained by unique complexities associated with the isolation of viable myocytes from the adult mouse heart. Current protocols rely on retrograde aortic perfusion using specialized Langendorff apparatus, which poses considerable logistical and technical barriers to researchers and demands extensive training investment. To identify and optimize a convenient, alternative approach, allowing the robust isolation and culture of adult mouse cardiac myocytes using only common surgical and laboratory equipment. Cardiac myocytes were isolated with yields comparable to those in published Langendorff-based methods, using direct needle perfusion of the LV ex vivo and without requirement for heparin injection. Isolated myocytes can be cultured antibiotic free, with retained organized contractile and mitochondrial morphology, transcriptional signatures, calcium handling, responses to hypoxia, neurohormonal stimulation, and electric pacing, and are amenable to patch clamp and adenoviral gene transfer techniques. Furthermore, the methodology permits concurrent isolation, separation, and coculture of myocyte and nonmyocyte cardiac populations. We present a novel, simplified method, demonstrating concomitant isolation of viable cardiac myocytes and nonmyocytes from the same adult mouse heart. We anticipate that this new approach will expand and accelerate innovative research in the field of cardiac biology.