心脏组织工程学研究的进展:成人体外心肌细胞原代与传代培养(英文)

目的:建立成人心肌细胞原代培养和传代培养的方法。方法:用0.2%胰蛋白酶(trypsin)和0.1%胶原酶(collagenase)进行消化,用差速黏附贴壁法纯化心肌细胞,用IMDM(Iscove's改良的Dul-becco'smedium)培养基培养,倒置显微镜下观察,用苔盼蓝(trypan)染色法做心肌细胞质量评价,心肌细胞进行Actin免疫组化、Myoglobin荧光免疫组化和电镜分析。结果:心肌细胞存活率为99%,24h后见有95%细胞贴壁,心肌细胞达95%,心肌细胞为圆形状、梭形状、椭圆状、棒状、星状及分叉状等;Actin免疫组化和Myoglobin荧光免疫组化强阳性,电镜见心肌细胞结构存在;原代培养第30天和传代培养第15天心肌细胞生长良好。结论:本方法可很好的用于成人心肌细胞原代培养和传代培养,为进一步建立心肌病理模型打好基础。

Progress of cardiac tissue engineering: primary and passaged culturing of cardiomyocytes from human adult ventricular myocardium in vitro

AIM:This study describes a method of isolating, culturing, purifying, subcultu ring, and identifying noncontracting ventricular myocytes grown from myocardium removed from valvular heart disease patients during corrective cardiac surgery. METHODS:Minced myocardium was digested in trypsin and collagenase. The isolate d cells were cultured in Iscove's modified Dulbecco's medium.The cardiomyocytes were purified by differential attachment technique. Subculturing was done by 0.1 25 % trypsin and 0.01 % ethylenediamine tetraacetic acid (EDTA) digestion an d transferring of the cells to new culture dishes containing the above culture m edium. The myocardial sarcomeric actin and myoglobin were identified by immunofl uorescence antibody staining. Myofibrils and mitochondria were visible by electr on microscopy. RESULTS:The ratio of viable cells was 99 % identified by trypan staining. Th e ratio of attachment cells was 95 % after 24 h in culture. The cultured human adult heart cells were roundness-shaped, rod-shaped, shuttle-shaped, ellipse -shaped, star-shaped and bifurcate-shaped with non-contractility. The myocar dial actin and myoglobin were identified by immunocytochemistry antibody stainin g and immunofluorescence antibody staining. The ultrastructure of cells was simi lar to that of the cardiac tissue in vivo by electron microscopy. Human adult he art cells after 30 d of primary culturing and after 15 d of passaged culturing w ere growed well.