Development of perfusion culture system of recombinant CHO cells for the production of urokinase-type Plasminogen Activator = Urokinase형 plasminogen activator를 생산하는 재조합 CHO 세포의 perfusion 배양계의 개발

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A serum-free medium with balanced amino acids was established for a high-density perfusion culture of a recombinant CHO cell line producing urokinase-type plasminogen activator (u-PA). Several protease inhibitors were examined for the prevention of significant conversion of scu-PA into tcu-PA, and a serine protease inhibitor, aprotinin was optimal at a concentration of 10 KIU ml. Serum-free or protein-free culture was proven to be more favorable for the prevention of the conversion of scu-PA than a serum-containing culture. A serum-free medium composed of IGF-I, transferrin, EGF and PDGF was constructed during the course of weaning cells to sequential reductions in FBS concentrations (5%, 3%, 2%, 1%, 0. 5% and 0.1%). In addition to the cells adapted to serum-free medium, cells adapted completely to a protein-free basal medium without any exogenous supplements except MTX and aprotinin was obtained. In tissue-flask cultures (25 ㎠), cells propagated to a density of $1.2×10^6$ cells/ml after 5 days in the serum-free medium, and their specific growth rate and specific productivity of u-PA were $0.026 h^{-1}$ and $134±58 IU/10^{-6}$ cells ㆍday, respectively. To balance nutrients in a perfusion medium, cellular requirements for amino acids were examined by a trial-and-error method, and a fortification of amino acids (aspartic acid, asparagine, serine, glutamine, threonine, arginine, valine, tryptophane, isoleucine, leucine, and methionine) was made. A control strategy for perfusion cultures was developed based on cellular consumption of oxygen and glucose and the perfusion performance was evaluated. Influences of microcarriers (solid and porous) and of different media (serum-free and serum-containing) were investigated. Using a solid microcarrier, cell growth and u-PA production were suppressed 2 ~ 3 times in the serum-free culture as compared with that in the control culture with 1% FBS medium. Using a porous microcarrier, cell growth and u-PA production were enha...
Kim, Jung-Hoeresearcher김정회researcher
한국과학기술원 : 생물과학과,
Issue Date
135091/325007 / 000935351

학위논문(박사) - 한국과학기술원 : 생물과학과, 1998, [ x, 125 p. ]


Serum-free medium; Spin-filter bioreactor; Perfusion culture; CHO cells; Microcarrier; 미립담체; 무혈청배지; 회전여과식 생물반응기; Perfusion배양; CHO 세포

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