磁性分离法高效快速培养原代C57BL/6J小鼠眼动脉平滑肌细胞

A magnetic separation method for rapid and efficient culture of primary C57BL/6J mouse ophthalmic artery smooth muscle cells

目的建立一种高效、快速地培养原代C57BL/6J小鼠眼动脉平滑肌细胞(OASMC)的磁性分离法。方法综合组织块贴壁法和酶消化法2种方法,通过优化胶原蛋白酶、琼脂糖氧化铁(Ⅱ,Ⅲ)磁性纳米粉末(以下简称铁粉)配方和铁粉粒径等关键实验环节,培养高纯度OASMC。原代OASMC培养和鉴定步骤:在无菌条件下,向小鼠左心室依次注入4℃PBS、琼脂糖铁粉混悬液,在解剖显微镜下分离出眼动脉。将其划至长为1.0~1.5 mm大小的组织块,用胶原蛋白酶消化45 min后,采用磁力分离器将眼动脉组织块从胶原蛋白酶中分离出来,均匀接种于培养皿中,置于37℃、5%CO_(2)条件下培养,待细胞生长至85%~90%时进行传代。对第3~7代细胞进行形态学及免疫荧光染色鉴定。结果通过改变不同条件确定了最佳实验条件:胶原蛋白酶与M199培养基比例为0.0015 g∶6 mL,胶原蛋白酶消化时间为45 min,铁粉粒径为5μm,琼脂糖与铁粉的比例为0.05 g∶0.05 g;琼脂糖铁粉混悬液的温度为37℃,注射器型号为0.6 mm×25 mm TWLB。原代培养2 d后可见眼动脉组织块周围有长梭形细胞爬出,呈放射状分布。培养7 d后,通过更换新的培养基铁粉逐渐减少,细胞生长速度加快,细胞形态呈现典型长梭形。免疫荧光染色结果显示所培养的OASMC表达α-平滑肌肌动蛋白。结论此方法培养原代C57BL/6J小鼠OASMC具有快速、高效、细胞纯度高等特点,为眼动脉相关疾病的药物活性筛选奠定了基础。

Objective To establish a magnetic separation method for rapid and efficient culture of the primary C57BL/6J mouse ophthalmic artery smooth muscle cells(OASMCs).Methods High purity OASMCs were cultured and identified by combining tissue block adhesion method and enzyme digestion method and optimizing the key experimental steps,including collagenase,the formula of agarose iron powder(Ⅱ,Ⅲ)and particle size of iron powder.The specific procedures were as follows:under sterile conditions,the left ventricle of mouse was successively injected with 4℃ phosphate buffered saline and agarose iron powder suspension,and the ophthalmic artery was isolated under dissecting microscope.It was cut into tissue blocks of 1.0-1.5 mm,digested with collagenase for 45 min,then separated from the collagenase by magnetic separator,planted evenly in a petri dish,and incubated at 37℃in an atmosphere of 5% CO_(2) in air.The OASMCs were subcultured when they reached 85%-90%confluence.The cells from 3 to 7 generations were identified by morphology and immunofluorescence staining.Results The optimal experimental conditions were determined by changing different conditions:the ratio of collagenase to M199 medium was 0.0015 g∶6 mL,the collagenase digestion time was 45 min,the particle size of iron powder was 5μm,and the mass ratio of agarose to iron powder was 0.05 g∶0.05 g;the temperature of the agarose iron powder suspension was 37℃,and the injector model was 0.6 mm×25 mm TWLB.After 2 d in primary culture,there were long spindle cells crawling out from the tissue blocks of the ophthalmic artery,with a radioactive distribution.After 7 d,iron powder was gradually reduced by replacing new medium,the speed of cell confluence was accelerated,and the cells showed a typical long spindle-shape morphology.Immunofluorescence staining of the cultured OASMCs demonstrated positiveα-smooth muscle actin.Conclusion The present method is rapid,efficient,and can achieve high purity cells for culturing primary C57BL/6J mouse OASMCs,which lays a foundation for screening drug activity of ophthalmic artery related diseases.