Plackett-Burman与响应面法相结合优化化学成分明确的MDCK细胞无血清无蛋白培养基

Optimization of chemically defined serum- and protein-free medium for MDCK cells by Plackett-Burman experiment design combined with response surface analysis

目的采用Plackett-Burman与响应面法相结合优化化学成分明确的MDCK细胞无血清无蛋白培养基。方法在自主研发的含蛋白无血清培养基的基础上,通过Plackett-Burman试验和响应面试验设计考察23组营养物质对MDCK细胞生长的影响,以细胞比生长速率、维持时间及最大活细胞密度作为响应值进行筛选和定量优化。以最优化的因子浓度配制化学成分明确的无血清无蛋白培养基,分别于细胞培养板和搅拌瓶中培养MDCK细胞,考察细胞比生长速率、维持时间和最大活细胞密度3个响应值的大小。结果通过Plackett-Burman试验筛选出4种对MDCK细胞生长具有显著性促进作用的因子:半胱氨酸、苏氨酸、亮氨酸和葡萄糖,苏氨酸和亮氨酸有助于提高细胞比生长速率,半胱氨酸和葡萄糖有利于提高最大活细胞密度;该化学成分明确的无血清无蛋白培养基能很好地支持MDCK细胞悬浮生长,单细胞悬浮培养MDCK细胞时,细胞比生长速率可达0.058/h,最大活细胞密度可达32.65×105个/ml,分别比基础培养基提高了2.15和3.09倍。结论采用Plackett-Burman与响应面法相结合优化了化学成分明确的MDCK细胞无血清无蛋白培养基,为采用MDCK细胞大规模工业化生产流感疫苗奠定了基础。

Objective To optimize chemically defined serum- and protein-free medium for MDCK cells by Plackett-Burman experiment design combined with response surface analysis. Methods For component screening and quantitative optimization, 23 groups of nutrients were investigated by the Plackett-Burman design and response surface analysis based on an in-house protein containing serum-free medium serving the specific growth rate, time duration and maximum viable cell density as response values. Chemical defined serum- and protein-free medium was formulated with the components at optimal concentrations, with which MDCK cells were cultured in cell culture plate and stirring bottle, and the response values were evaluated. Results Four factors promoting the growth of MDCK cells significantly, cysteine, threonine, leucine and glucose, were screened by Plackett-Burman test. Threonine and lecucine were helpful to increasing the specific cell growth rate, while cysteine and glucose to increasing maximum viable cell density. The chemical defined serum-free and protein-free medium supported the suspending growth of MDCK cells effectively. The specific growth rate and maximum viable cell density of MDCK cells in single-cell suspension culture reached 0. 058/h and 32. 65 × 10^5 cells/ml, which are 2. 15 and 3. 09 times higher than those in basal medium, respectively. Conclusion The chemically definedserum- and protein-free medium for MDCK cells was optimized by Plackett-Burman experiment design combined with response surface analysis, which laid a foundation of industrial production of influenza vaccine by using MDCK cells.