Vero细胞无血清培养基的优化  

Optimization of serum-free medium for Vero cells

作　　者：段盼盼[1] 杨帆[1] 耿兴良 龙云凤[1] 郭琦[1] 龙艺[2] 王明清[1] 廖国阳[1] 李卫东[1] 

机构地区：[1]中国医学科学院北京协和医学院医学生物学研究所云南省重大传染病疫苗研发重点实验室,云南昆明650118 [2]苏州大学医学部基础医学与生物科学学院,江苏苏州215123

出　　处：《中国生物制品学杂志》2015年第5期518-522,共5页Chinese Journal of Biologicals

基　　金：863计划腮腺炎病毒疫苗等病毒性疫苗关键技术及产品开发(2012AA02A404);科技部国际合作项目Sabin IPV关键技术研究(2010DF32890)

摘　　要：目的通过试验设计(design of experiments,DOE)方法和统计学分析快速优化Vero细胞无血清培养基。方法以DMEM/F12为基础培养基,通过Plackett-Burman试验、最陡爬坡试验和Box-Behnken试验考察7种添加物胰岛素、人转铁蛋白、透明质酸、牛血清白蛋白、亚硒酸钠、表皮生长因子和高密度脂蛋白对Vero细胞生长的影响,以CCK-8测定的吸光度值(A490)为响应值,优化Vero细胞无血清培养基。用最优化的因子浓度配制的Vero细胞无血清培养基培养Vero细胞,对优化结果进行验证。结果通过Plackett-Burman试验筛选出胰岛素、牛血清白蛋白和人转铁蛋白对Vero细胞生长影响较大;采用最陡爬坡试验和Box-Behnken试验进一步优化,Design-Expert 8.06软件进行回归分析,得到胰岛素、人转铁蛋白和牛血清白蛋白的最佳浓度分别为5.625μg/ml、14.034μg/ml和3.92 mg/ml,在此优化条件下的A490值为2.3,较基础培养基提高了3.41倍。用最优的胰岛素、人转铁蛋白和牛血清白蛋白浓度配制的Vero细胞无血清培养基培养Vero细胞的A490值为2.148,为预测值的93.4%,符合度较高。结论应用DOE方法快速高效地优化了Vero细胞无血清培养基,为无血清培养基的研制奠定了基础。Objective To rapidly optimize a serum-free medium for Vero cells by design of experiment（DOE） and statistical analysis. Methods Using DMEM / F12 as basal medium, the effect of seven supplements, i. e. insulin, human transferrin, hyaluronic acid, bovine serum albumin（BSA）, sodium selentite, epidermal growth factor（EGF） and high density lipoprotein（HDL）, on the growth of Vero cells were investigated by Placket-Burman design, the steepest ascent experiment and Box-Behnken design based on A490 values tested by CCK-8 as a response value. Vero cells were cultured by the optimized serum-free medium to verify the optimization efficacy. Results Factor screening analysis by PlackettBurman design revealed that insulin, human tranferrin and BSA influenced the growth of Vero cells significantly. Further optimization by the steepest ascent experiment and Box-Behnken design and regression analysis by Design-Expert 8. 06 software that the optimal concentrations of the three supplements were 5. 625 μg / ml, 14. 034 μg / ml and 3. 92 mg / ml respectively. The A490 value under the optimal condition was 2. 3, which was 3. 41 times higher than that in basal medium.The A490 value of Vero cells cultured in the optimized medium was 2. 148, which was 93. 4% of that expected. Conclusion A serum-free medium for Vero cells was optimized effectively by DOE, which laid a foundation of development of serumfree medium.

关 键 词：VERO细胞 无血清培养基 优化 试验设计 

分 类 号：R392-33[医药卫生—免疫学]