胚胎期及出生后不同年龄段小鼠心肌细胞体外分离方法及优化

Optimization of Isolation Methods of Cardiomyocytes From Embryonic and Postnatal Mice of Different Ages

目的:基于目前已有报道的心肌细胞体外消化及分离方法,对胚胎期及出生后不同年龄段小鼠心肌细胞体外分离的方法进行系统探索和优化。方法:取胚胎期及出生后不同年龄段小鼠心脏,运用不同方法分离心肌细胞。利用Count Star仪器检测心肌细胞大小及活性。取细胞悬液涂片,针对心肌细胞标志物α辅肌动蛋白(α-actinin)表达进行免疫荧光染色,用共聚焦显微镜观察心肌细胞形态。结果:用胰酶消化法分离胚胎期小鼠心肌细胞,获得存活率大于94%的心肌细胞。用心脏解离试剂盒法(Gentle MACS法)体外分离新生小鼠(出生后1~3 d)心肌细胞,获得存活率约为97%的心肌细胞。分别用Gentle MACS法、非Langendorff灌流法分离出生后4 d和7 d的小鼠心肌细胞,前者仅获得存活率为58%的心肌细胞,而后者获得的心肌细胞存活率为70%~80%。对于成年小鼠,非Langendorff灌流法和Langendorff灌流法均可获得杆状率、存活率都在70%左右的心肌细胞。结论:结合文献以及本实验结果发现,分离胚胎期小鼠心肌细胞可用胰酶消化法;分离出生后1~3 d小鼠心肌细胞可用Gentle MACS法。对于出生后4 d和7 d的小鼠,非Langendorff灌流法可获得存活率及杆状率均较高的心肌细胞,分离成年小鼠心肌细胞则适合用非Langendorff灌流法和Langendorff灌流法。

Objectives:Based on the reported methods of in vitro digestion and isolation of cardiomyocytes,the methods of in vitro isolation of cardiomyocytes from embryonic and postnatal mice of different ages were systematically explored and optimized.Methods:Embryonic and postnatal mouse hearts of different ages were excised and cardiomyocytes were isolated using different methods.The size and activity of isolated cardiomyocytes were measured using the Count Star equipment.Then the cell suspension was smeared and the cardiomyocyte markerα-actinin was used for immunofluorescence staining to observe the morphology of cardiomyocytes under the confocal microscope.Results:The isolation approach of mouse cardiomyocytes at embryonic stage with the trypsin digestion method achieved a survive rate of more than 94%.The isolation approach of cardiomyocytes in neonatal mice(1-3 days after birth)with the Gentle MACS method achieved a survive rate of more than 97%.The isolation approach of mouse cardiomyocytes at 4 and 7 days after birth with the Gentle MACS achieved a survival rate of 58%,and non-Langendorff perfusion method achieved a survival rate of 70-80%.For adult mice,cardiomyocytes with rod-shape rate and survival rate of about 70%could be achieved by both the non-Langendorff perfusion and Langendorff method.Conclusions:According to the previous reports and our experimental results,we find that the best method for isolating mouse cardiomyocytes in embryonic stage is the trypsin digestion approach;the superior method for isolating mouse cardiomyocytes at 1 to 3 days after birth is the Gentle MACS method.For mice at 4 and 7 days after birth,non-Langendorff perfusion method could be the good choice to obtain cardiomyocytes with higher survival rate and rodshaped rate,and both non-Langendorff perfusion method and Langendorff method could be used for isolating mouse cardiomyocytes of adult mice.