Nag A C, Cheng M, Fischman D A, Zak R
J Mol Cell Cardiol. 1983 May;15(5):301-17. doi: 10.1016/0022-2828(83)91342-1.
Adult rat heart was dissociated into a single-cell suspension by a retrograde perfusion technique with collagenase and hyaluronidase in Krebs-Ringer phosphate buffer. Long-term culture of these isolated single cardiac muscle cells was established for up to 45 days. Transmission electron microscopy and immunofluorescence analysis with monoclonal antibodies to cardiac myosin were used to examine sequentially the external and internal structural organization of the cardiac myocytes. Most of the cardiac myocytes exhibited prominent alterations in their external and internal structural organization during the first two weeks of culture. As they attached to the substrate and spread out, the myocytes assumed various shapes and sizes, with the exception of a few which maintained their original cylindrical shape. Electron microscopy of 2 to 4-day cultures revealed that most of the muscle cells contained disorganized myofibrils and surface blebs with enclosed mitochondria and myofilaments, which were eventually extruded from the cytoplasm. With progressive culture, the cardiac myocytes appeared to lose myofibrillar material; fewer myofilaments or sacromere fragments with interfibrillar mitochondria were observed in the sarcoplasm. Such cells resembled cultured embryonic or neonatal cardiac myocytes. However, some muscle cells retained closely packed, well organized myofibrils characteristic of freshly dissociated or in vivo cardiac myocytes. Immunofluorescence microscopy demonstrated that the cultured cardiac myocytes were strongly myosin positive throughout their morphological changes and subsequent maintenance in culture. Two patterns of fluorescence were observed in these cells in correlation with the fine structural evidence for myofibrillar distribution. One pattern exhibited bright fluorescence near the central region of the cell with a more weakly diffuse fluorescence throughout the cytoplasm; the other pattern was characterized by bright fluorescence throughout the sarcoplasm. Most of the myocytes retained their contractility throughout the culture period excepting the initial 24 to 48 h of cell attachment and flattening. These studies demonstrate the feasibility of maintaining contractile cardiac muscle cells from adult rats for at least 1 1/2 months in monolayer culture, although some variability in myofibrillar organization has been observed.
成年大鼠心脏通过在磷酸缓冲的克氏液中用胶原酶和透明质酸酶进行逆行灌注技术解离成单细胞悬液。这些分离的单个心肌细胞进行了长达45天的长期培养。使用透射电子显微镜和针对心肌肌球蛋白的单克隆抗体进行免疫荧光分析，依次检查心肌细胞的外部和内部结构组织。大多数心肌细胞在培养的前两周内其外部和内部结构组织出现显著改变。当它们附着于底物并铺展时，心肌细胞呈现出各种形状和大小，只有少数保持其原来的圆柱形。对培养2至4天的细胞进行电子显微镜检查发现，大多数肌肉细胞含有排列紊乱的肌原纤维和表面泡，泡内含有线粒体和肌丝，这些最终从细胞质中挤出。随着培养的进行，心肌细胞似乎失去了肌原纤维物质；在肌浆中观察到较少的肌丝或肌节片段以及肌原纤维间的线粒体。这样的细胞类似于培养的胚胎或新生心肌细胞。然而，一些肌肉细胞保留了紧密排列、组织良好的肌原纤维，这是刚解离的或体内心肌细胞的特征。免疫荧光显微镜显示，培养的心肌细胞在其形态变化及随后的培养维持过程中始终呈强烈的肌球蛋白阳性。在这些细胞中观察到两种荧光模式，与肌原纤维分布的精细结构证据相关。一种模式在细胞中央区域附近呈现明亮荧光，细胞质中荧光较弱且呈弥漫状；另一种模式的特征是整个肌浆呈现明亮荧光。除了细胞附着和平展的最初24至48小时外，大多数心肌细胞在整个培养期间都保持其收缩性。这些研究证明了在单层培养中使成年大鼠的收缩性心肌细胞维持至少1个半月的可行性，尽管已观察到肌原纤维组织存在一些变异性。
