Cultivation of recombinant Chinese hamster ovary cells grown as suspended aggregates in stirred vessels.
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 mum 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 (mu). 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-l stirred tank bioreactor for 15 d resulted in a maximum viable cell density of 5.6 x 10(6) cells ml(-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.
Han
Yi
Y
Department of Cell Engineering, Beijing Institute of Biotechnology, 20 Dongdajie Street, Fengtai, Beijing 100071, P.R. China.
Liu
Xing-Mao
XM
Liu
Hong
H
Li
Shi-Chong
SC
Wu
Ben-Chuan
BC
Ye
Ling-Ling
LL
Wang
Qu-Wei
QW
Chen
Zhao-Lie
ZL
D000818:Animals; D019149:Bioreactors; D016466:CHO Cells / Q000166:cytology*; D002449:Cell Aggregation; D018929:Cell Culture Techniques / Q000379:methods*; D049109:Cell Proliferation; D048271:Chitosan / Q000494:pharmacology; D006224:Cricetinae; D003470:Culture Media; D011995:Recombination, Genetic; D013535:Suspensions
D003470:Culture Media; D013535:Suspensions; D048271:Chitosan