原代乳鼠心肌细胞分离培养的改进

An improved protocol for primary cultur of myocytes extracted from neonatal rat

目的探讨提高细胞纯度、存活率及活性的乳鼠心肌分离培养方法。方法改进心脏组织的处理方法,用胰酶预处理后再用II型胶原酶依次处理心肌组织,采用差速贴壁及化学药物纯化心肌细胞。结果提取的单个心肌细胞结构完整,呈类圆形;12 h时大部分细胞贴壁;24 h后几乎全部贴壁,仅见少量漂浮细胞,贴壁细胞伸出伪足,呈梭形、三角形等,细胞出现自主搏动。48 h后心肌细胞伪足间形成联接,交织成网状,局部心肌细胞出现同步搏动。3 d后心肌细胞聚集成簇,呈岛屿样搏动。心肌细胞存活率为96.6%,纯度>96%。结论此改进方法所提取培养的乳鼠心肌细胞存活率及纯度高,细胞活性好,结构功能完整,可用于相关实验中原代乳鼠心肌细胞的分离培养。

Objective To improve the traditional method of culturing neonatal rat myocytes for the better purity,survival rate and activity of myocytes in experiments.Methods Trypsin and type Ⅱ collagen enzyme were used to digest heart tissue and myocytes were purified with differential sticking wall method and chemical purification.Results The extracted single myocyte is of structural integrity and circular shape.Most cells were touching to wall in 12 hours,and all cells sticking to wall in 24 hours,with small amount of floating cells.The touching-wall cells extended pseudopodia in the form of fusiformis,triangle and so on,with independent rhythm.Myocardial cells＇ pseudopodia became connected into webs 48 hours later,with local synchronization rhythm.There days later,myocytes gethered into clusters,with island-like pulse.The activity and purity of myocytes was up to 96%.Conclusion This method helped improve the activity and purity of myocytes,and protect the integrity of cells.The extracted myocytes could be used in relative experiments.