Isolation and Cultivation of Adult Rat Cardiomyocytes
Isolation and Cultivation of Adult Rat Cardiomyocytes
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DOI：
10.3791/56634
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发表时间：
2017-10-01
影响因子：
1.2
通讯作者：
Schlueter, Klaus-Dieter
中科院分区：
文献类型：
--
作者：
Nippert, Franziska;Schreckenberg, Rolf;Schlueter, Klaus-Dieter
在完整的心脏中，相邻细胞影响成年心肌细胞。通过分离和培养成体心肌细胞的方法，可以精确研究这些细胞在特定处理和环境下的行为。本手稿提出了成功分离和培养成年大鼠心室心肌细胞（ARVC）的方案。在深度麻醉下通过颈椎脱位处死大鼠。然后，取出心脏并暴露主动脉。随后，在 Langendorff 灌注系统上进行钙消耗和胶原酶处理的灌注。然后，心室组织被切碎、再循环和过滤，然后进行三个离心步骤，并逐渐添加 CaCl2，直至达到生理钙浓度。 ARVC 铺在细胞培养皿上。更新细胞培养基后，ARVC 可以在不更换含血清培养基的情况下培养长达六天。 ARVC 的分离是一个钙敏感过程。细胞内钙浓度的微小变化会导致分离细胞的质量和活力下降。新鲜分离的 ARVC 呈棒状。在培养的最初几天内，它们失去了杆状形态并形成伪足状结构（传播）。在此形态形成过程中，ARVC 最初降解其收缩元件，然后通过肌动蛋白应力纤维和从头肌瘤发生进行重组。培养一周后，大多数 ARVC 表现出广泛的外观，并具有清晰可检测的交叉条纹。该过程对细胞内钙浓度敏感，因为用离子霉素治疗会减弱扩散。这一去分化和再分化过程中的关键标志物是 β-肌球蛋白重链 (β-MHC)、制瘤素 M (OSM) 和 swiprosin-1 (EFHD2)。最近的研究表明，培养条件下发生的心脏再分化和去分化模仿了心脏重塑过程中体内观察到的特征。因此，ARVC的分离和培养对于理解心肌细胞的生物学起着关键作用。
In an intact heart, adjacent cells influence adult cardiomyocytes. With the method of isolation and cultivation of adult cardiomyocytes, a precise investigation of the behavior of these cells under specific treatments and environments is possible. This manuscript presents a protocol for successful isolation and cultivation of adult rat ventricular cardiomyocytes (ARVC).The rat is sacrificed by cervical dislocation under deep anesthesia. Then, the heart is extracted and the aorta is uncovered. Subsequently, perfusion on the Langendorff perfusion system with calcium depletion and collagenase treatment is performed. Afterwards, ventricular tissue gets minced, re-circulated, and filtered, followed by three centrifugation steps with gradual addition of CaCl2 until physiological calcium concentration is reached. ARVC are plated on cell culture dishes. After refreshing the cell culture medium, ARVC can be cultivated for up to six days without changing the serum-containing culture medium. Isolation of ARVC is a calcium sensitive process. Small changes in the intracellular calcium concentration cause a decrease in the quality and viability of the isolated cells.Freshly isolated ARVC are rod shaped. Within the first days of cultivation they lose the rod-shaped morphology and form pseudopodia-like structures (spreading). During this morphological formation ARVC initially degrade their contractile elements followed by a reformation through actin stress fibers and de novo sarcomerogenesis. After one week of cultivation, most ARVC show a widespread appearance with a clearly detectable cross striation. This process is sensitive to intracellular calcium concentration, as treatment with ionomycin attenuates spreading. Key markers in this process of de- and re-differentiation are beta-myosin heavy chain (beta-MHC), oncostatin M (OSM), and swiprosin-1 (EFHD2). Recent studies have suggested that cardiac re- and de-differentiation occurring under culture conditions mimics features seen in vivo during cardiac remodeling. Therefore, isolation and cultivation of ARVC play a key role in understanding the biology of cardiomyocytes.