서지주요정보
High cell density continuous culture of anchorage-dependent animal cells in a cylindrical depth filter perfusion system = 원통형 깊이필터를 이용한 부착성 동물세포의 고농도 연속배양
서명 / 저자 High cell density continuous culture of anchorage-dependent animal cells in a cylindrical depth filter perfusion system = 원통형 깊이필터를 이용한 부착성 동물세포의 고농도 연속배양 / Sang-Kyo Choi.
저자명 Choi, Sang-Kyo ; 최상교
발행사항 [대전 : 한국과학기술원, 1995].
Online Access 원문보기 원문인쇄

소장정보

등록번호

8005934

소장위치/청구기호

학술문화관(문화관) 보존서고

DCHE 95018

SMS전송

도서상태

이용가능

대출가능

반납예정일

초록정보

A depth filter perfusion system (DFPS) equipped with cylindrical depth filter was applied to high cell density continuous culture of anchorage-dependent animal cells. Whole cell enzyme immobilization using a depth filter was also investigated as an application of the DFPS. Cylindrical depth filters are widely used in industries handling fluids containing solid particles. Many household water purification systems also use these cylindrical depth filters which are disposable and inexpensive, and have good filtration performances. An important feature of depth filters is that captured particles are distributed homogeneously in the filter matrix, which improves particle removal efficiency and makes long-term operation possible without filter clogging. The DFPS filter units are available commercially in large quantities at a reasonable price and they are also uniform in quality. In addition, the DFPS provides advantages of high surface-to-volume ratio of 450-600 ㎠/㎤, low cost set-up, easy operation and scale-up. To identify the basic characteristics of the DFPS, residence time distributions, surface modifications by coating various materials and oxygen transfer properties by bubble aeration were investigated. From the analysis of residence time distributions, the DFPS exhibits a plug flow behavior with some dispersion. In the 20 and 40 ㎛ filter, increase of the circulation rate did not affect significantly to the mixing patterns. However, in the 70 ㎛ filter, dispersion of the fluid decreased as increasing the circulation rate. Surface modification by gelatin coating increased the cation and anion exchange capacities of the depth filter and promoted the attachment rate of the cells tested onto the filter surfaces. Pluronic F-68 containing medium and sparging with the micron-size filter could supply enough oxygen to support a cell concentration in the order of $10^8$ cells/ml, theoretically. Vero cells and recombinant CHO cells were cultivated in the DFPS to test the feasibility of using DFPS for high density cultures of anchorage-dependent cells. In the Vero cell cultures, gelatin coating on polypropylene fibers in the DFPS was necessary to promote the cell attachment and growth. Dissolved oxygen (DO) concentrations could be controlled by sparging air into the reservoir vessel through a filter sparger. When DO concentration was controlled above 40% of air saturation in the DFPS with 40 mm pore size, the maximum viable cell concentration estimated was $3.81×$10^7$ cells/ml of the total reactor volume. This viable cell concentration is approximately 18 times higher than that obtained in a T-flask batch culture. Recombinant CHO cells producing t-PA were also successfully cultivated in the DFPS with high cell density and stable operations. Dissolved oxygen concentrations could be controlled at 40% air saturation by sparging air into the reservoir vessel with a micron size stainless steel filter sparger. The DFPS supported a high cell concentration of $.1.8×$10^7$ cells/ml based on the total reactor volume. The produced t-PA in the DFPS was a good quality during the whole culture periods. The depth filter can be used for cell separation from culture broth, immobilization, and whole cell enzyme reaction for any kind of microorganisms because the filter has a broad specification of the pore sizes. Therefore, more than 3 steps among the overall production processes using whole cell enzymes can be simplified to one step by using the depth filter unit. To test the possibility of using DFPS as a whole cell enzyme reactor, whole microbial cell enzymes of Brevibacterium sp. CH2 (nitrile hydratase) and recombinant Saccharomyces cerevisiae (invertase) were immobilized in cylindrical depth filters with different pore sizes. The immobilized whole cell enzymes were used to produce high quality acrylamide from acrylonitrile and glucose and fructose from sucrose in a fed-batch and continuous mode. In this work, immobilization of Brevibacterium sp. CH2 and the yeast cells without significant pressure drop was successfully performed using the depth filter having a pore size of 0.5 and 5 ㎛, respectively. In the fed-batch operations using the immobilized whole cell enzymes, the acrylamide concentration of 150 g/L and glucose concentration of 375 g/L were obtained. These values were slightly higher than that in the resting cells. In the continuous operation with the depth filter unit, high glucose or fructose productivity of 68 g/L/h was obtained and steady state operation for a long period was possible. In the depth filter immobilization unit, mass transfer was not a serious problem because forced convection could be achieved by fast circulation of the reaction mixture. Therefore, the rate limiting step is not the mass transfer in the depth filter unit but the reaction by enzymes. Accordingly, the DFPS displayed its potential for being one of the promising bioreactor systems for anchorage-dependent animal cell cultures and whole cell enzyme reactions.

서지기타정보

서지기타정보
청구기호 {DCHE 95018
형태사항 xix, 158 p. : 삽도 ; 26 cm
언어 영어
일반주기 저자명의 한글표기 : 최상교
지도교수의 영문표기 : Ho-Nam Chang
지도교수의 한글표기 : 장호남
학위논문 학위논문(박사) - 한국과학기술원 : 화학공학과,
서지주기 Reference : p. 140-150
주제 Depth Filter Perfusion System
Animal Cell Culture
Vero
CHO
Whole Cell Enzyme Immobilization
Invertase
Nitrile Hydratase
깊이필터
동물세포배양
베로 세포
초 세포
전세포 효소 고정화
인버타제
니트릴하이드라타제
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