多孔微载体无血清培养rCHO细胞生产u-PA  

Production of u-PA with rCHO Cell Culture on Porous Microcarriers in Serum free Growth Medium

作　　者：胡显文[1] 肖成祖[1] 李佐虎[2] 郭志霞[1] 高丽华[1] 张正光[1] 胥照平[1] 王菲[1] 

机构地区：[1]军事医学科学院生物工程研究所,北京100071 [2]中国科学院化工冶金研究所生化工程国家重点实验室,北京100080

出　　处：《生物工程学报》2000年第3期387-391,共5页Chinese Journal of Biotechnology

基　　金：国家高技术研究发展计划项目资助!(Z18 0 3 12 )

摘　　要：在 30L搅拌式反应器中无血清培养分泌尿激酶型纤溶酶原激活剂 (u PA)的DNA重组CHO细胞 ,定期部分更换Cytopore多孔微载体 ,使生长在多孔微载体中的细胞不断更新繁殖 ,解决大规模细胞培养中的细胞凋亡问题。在 91d连接换液培养过程中 ,细胞密度可维持在 (1 3～ 2 6 )× 10 7/mL ,活细胞比率维持在 90 %以上。在7 5L搅拌罐中培养细胞 ,利用外部周期性压力振荡刺激并结合载体更新技术 ,可减轻密度效应对细胞生长和表达的影响 ,在一定程度上提高细胞在高密度培养条件下的表达水平。在 6 7d连续换液培养中 ,细胞最高密度为 2 6 4× 10 7/mL ,活细胞比率维持在 95 %以上。与稳压操作相比 ,利用周期变压刺激技术可提高产量 10 %～ 2 0 % ,且可降低葡萄糖厌氧代谢生成乳酸的转化率 ,利用 4步纯化工艺 ,从含u PA约 135g的 2 10 0L上清中获得约 80 gu PA(单链比例约为 90 % )。A novel technique was developed to deal with apoptosis in large scale animal cell culture.By means of replacing part of Cytopore porous microcarriers at regular intervals,a rCHO cell line,which produces urokinase type plasminogen activitor(u PA),was cultivated continuously with serum free medium in a 30L stirred tank for 91 days.The cell density was maintained at (1.3～2.6)×10 7/mL,and >90% of cells was viable.In order to reduce the effect of cell density on cell growth and expression,a cyclic pressure oscillation was exerted on a 7.5L reactor headspace to enhance cell expression at high cell density to a certain extent.During the 67 days of medium replacement culture,the maximal cell density reached 2.64×10 7/mL,and cell viability was always kept above 95% when combined with microcarrier replacement.Compare to control culture,culture with cyclic pressure oscillation could enhance cell expression level and reduce the ratio of glucose metabolized anaerobically to produce lactate.With four step purification process,about 80 g u PA(～90% scu PA)was recovered from ～2100 liters supernatant which contained～135 g u PA.

关 键 词：动物细胞培养 微载体更新 U-PA RCHO细胞 

分 类 号：Q813.11[生物学—生物工程]