适于重组CHO细胞培养的无血清培养基的制备  

Preparation of Serum-free Medium Suitable for Culture of CHO Cells

作　　者：张大鹤[1] 易小萍[1] 张元兴[1] 孙祥明[1] 

机构地区：[1]华东理工大学生物反应器工程国家重点实验室,上海200237

出　　处：《中国生物制品学杂志》2011年第10期1152-1156,共5页Chinese Journal of Biologicals

摘　　要：目的制备适于重组CHO细胞培养的无血清培养基(Serum-free medium,SFM)。方法在本室制备的无血清、低蛋白培养基SFMC的基础上,开发支持重组CHO-K1细胞生长的无动物源、无蛋白培养基(Protein-free midium,PFM,以柠檬酸铁、硫酸锌和酵母水解物替换SFMC中的转铁蛋白、胰岛素和动物组织水解物)及化学成分确定的培养基(Chemically definedmedium,CDM,以PFM培养基为基础,通过调整和优化氨基酸的浓度和其他部分营养物质浓度制备),评价各种无血清培养基的适应性、稳定性以及冻存、复苏性能,并分别在125 ml摇瓶和1.4 L生物反应器中进行培养。结果 CHO-K1细胞在PFM和CDM中生长良好,经125 ml摇瓶培养,最高细胞密度分别为9.3×106和7.3×106个/ml,最大细胞密度与SFMC培养基相比,分别提高了107%和62%。经1.4 L搅拌式生物反应器流加培养,PFM培养基的最高细胞密度可达9.8×106个/ml,培养216 h时,细胞活性仍维持在80%,重组蛋白表达量达110.5 mg/L;采用CDM培养基经流加培养,CHO-K1细胞最大密度可达9.5×106个/ml,但细胞培养时间较PFM培养基短。结论在SFMC培养基的基础上,成功开发出培养效果更优的PFM和CDM培养基。Objective To prepare the serum-free medium suitable for culture of CHO cells.Methods Non-animal-derived protein-free medium（PFM） supporting the growth of recombinant CHO cells was developed by substituting the transferring,insulin and animal tissue lysate in SFMC,a serum-free medium prepared by the authors,with ferric citrate,zinc sulfate and yeast lysate respectively,based on which chemically defined medium（CDM） was developed by optimizing the concentrations of amino acids and partial other nutrient substances in PFM.Both PFM and CDM were evaluated for suitability,stability as well as frozen and resuscitation performances,and cultured in 125 ml shake flask and 1.4 L bioreactor.Results CHO-K1 cells grew well in both PFM and CDM.After culture in 125 ml shake flask,the maximum densities of CHO-K1 cells in PFM and CDM were 9.3 × 106 and 7.3 × 106 cells / ml,which increased by 107% and 62% as compared with those in SFM respectively.However,after fed-batch culture in 1.4 L stirring bioreactor,the maximum density of CHO-K1 cells in PFM reached 9.8 × 106 cells / ml.Meanwhile,the activity of cells was still 80% after culture for 216 h,and the expression level of recombinant protein reached 110.5 mg / L.The maximum density of CHO-K1 cells after fed-batch culture in CDM reached 9.5 × 106 cells / ml,while the time for culture was shortened compared with that in PFM.Conclusion PFM and CDM which were more suitable for culture of CHO cells were successfully developed on the basis of SFMC.

关 键 词：CHO细胞 培养基 无血清 

分 类 号：R392-33[医药卫生—免疫学]