Optimization of yeast-derived serum-free medium by Plackett-Burman design and response surface methodology
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 × 106 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.
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