Cultivation of recombinant Chinese hamster ovary cells grown as suspended aggregates in stirred vessels
Cultivation of recombinant Chinese hamster ovary cells grown as suspended aggregates in stirred vessels
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DOI：
10.1263/jbb.102.430
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发表时间：
2006-11-01
影响因子：
2.8
通讯作者：
Chen, Zhao-Lie
中科院分区：
文献类型：
--
作者：
Han, Yi;Liu, Xing-Mao;Chen, Zhao-Lie
描述了能够产生尿激酶原突变体(MPRO-UK)的重组中国仓鼠卵巢(RCHO)细胞，在搅拌容器中以悬浮聚集体的形式生长。在DMEM和Ham‘s F12(D-MEM/F-12)混合培养基中添加壳聚糖能有效促进细胞聚集和球体的形成。RCHO细胞接种到添加壳聚糖的培养液中后立即形成多细胞聚集体，反映聚集体大小的细胞聚集体的平均直径随着培养时间的延长而增加，在转瓶中培养2d和9d后，细胞聚集体的平均直径从65 pm移动到163 pm。悬浮聚集体和锚定单层的rCHO细胞的代谢性能没有显著差异。然而，从比生长率(A)来看，以悬浮聚集体培养的细胞生长速度明显下降。用CD293替代D-MEM/F-12培养基使致密的球形细胞聚集体解离成小的不规则聚集体和单个细胞，对继代培养的细胞性能没有明显影响。在2-1搅拌式生物反应器中以悬浮聚集体形式生长的rCHO细胞灌流培养15d，最大活细胞密度为5.6×10(6)个细胞/(-1)，MPRO-UK浓度约为2.6×10(3)IU/(-1)，细胞存活率在整个运行过程中保持在约90%。
Recombinant Chinese hamster ovary (rCHO) cells capable of producing a prourokinase mutant (mPro-uk) grown as suspended aggregates in stirred vessels were described and characterized. The addition of chitosan to a mixture of DMEM and Ham's F12 (D-MEM/F-12) medium promoted cell aggregation and spheroid formation efficiently. Multicellular aggregates formed immediately after the rCHO cells were inoculated into the chitosan-added medium, and the mean diameter of the cell aggregates reflecting the aggregate size increased with culture time, shifting from 65 to 163 pm after 2 and 9 d of culture in spinner flasks. No significant difference in the metabolism performance of the rCHO cells was observed between suspended aggregates and anchored monolayers. However, the cells cultured as suspended aggregates showed a marked decrease in growth rate as evaluated from specific growth rate (A). Replacing D-MEM/F-12 medium with CD 293 medium caused compact spherical cell aggregates to dissociate into small irregular aggregates and single cells without apparent effects on cell performance in subcultures. The perfusion culture of the rCHO cells grown as suspended aggregates in a 2-1 stirred tank bioreactor for 15 d resulted in a maximum viable cell density of 5.6 x 10(6) cells mI(-1) and an mPro-uk concentration of about 2.6 x 10(3) IU ml(-1), and cell viability was remained at roughly 90% during the entire run.