Plackett-Burman实验和响应面法优化酵母源无血清培养基

Optimization of yeast-derived serum-free medium by Plackett-Burman design and response surface methodology

目的采用Plackett-Burman实验和响应面法(response surface methodology,RSM)优化酵母源无血清培养基(serum-free medium,SFM)的营养组分,明确培养基成分及浓度。方法以中国仓鼠卵巢细胞(CHO)为研究对象,在实验室前期筛选出的SFM的基础上,采用Plackett-Burman实验对10种营养组分进行筛选。筛选出对细胞具有促生长作用的因素后,利用RSM进行二次优化,以阐明各变量间的相互作用,确定促CHO细胞生长的最佳培养基组成。取对数生长期CHO细胞,用优化后的培养基重悬后,进行传代扩增。结果经Plackett-Burman实验筛选的10个变量中,酵母抽提物(yeast extract,YE)FM888和微量元素能有效提高细胞比生长速率;非必需氨基酸和FM888对提高细胞存活天数作用显著;FM888、必需氨基酸、非必需氨基酸及微量元素能显著提高活细胞密度。综合选择FM888、微量元素和非必需氨基酸3个因素进行RSM优化试验。优化后的培养基悬浮培养CHO细胞时,其最大比生长速率可达0.025/h,存活天数可达6 d,最大活细胞密度可达7.187×106个/mL。将优化后的培养基进行细胞稳定传代试验,可稳定传代12代,其活细胞密度及细胞活率与对照组(商业化SFM)接近。结论成功获得成分明确且能支持CHO细胞高密度生长的酵母源SFM,为YE在动物细胞培养中的应用以及高效SFM的开发提供了参考和依据。

Objective To optimize the nutrient components of yeast-derived serum-free medium(SFM)and determine the composition and concentration by Plackett-Burman design and response surface methodology(RSM).Methods Based on the SFM selected in the early stage of experiment,10 nutrient components were screened by PBD,using Chinese hamster ovary(CHO)cells as the research object,which were optimized secondarily by RSM to elucidate the interaction among the variables and determine the best medium composition for the growth of CHO cells,after the factors promoting the growth of CHO cells were screened out.CHO cells in logarithmic growth stage were re-suspended in the optimized culture medium,and then subcultured and amplified.Results Among the 10 variables screened by Plackett-Burman experiment,yeast extract(YE)FM888 and trace elements effectively increased the specific growth rate of cells;Non-essential amino acids and FM888 showed significant effects on improving cell survival days;FM888,essential amino acids,non-essential amino acids and trace elements significantly increased the density of viable cells.Based on this,three factors,FM888,trace elements and non-essential amino acids,were comprehensively selected for RSM optimization test.When cultured in the optimized medium in suspension,the maximum specific growth rate of CHO cells was 0.025/h,the survival days were 6 d,and the maximum viable cell density was 7.187×10°cells/mL.The optimized medium was used for cell passage test,based on which,the cells were stably passaged for 12 passages,and the viable cell density and cell viability were close to those of the control group(commercial SFM).Conclusion The yeast-derived SFM with clear components and supporting the high-density growth of CHO cells was obtained,which provided a reference and basis for the application of YE in animal cell culture and the development of high-efficiency SFM.