• KSBB Journal
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• 제30권3호
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• pp.103-108
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• 2015

Production of foreign proteins by transgenic plant cell cultures has several advantages such as post-translational modification, low risk of product contamination and low-cost production and purification. However, target proteins are degraded by extracellular proteases existing in the media. A solution to this problem is the use of perfusion culture and ion exchange chromatography for the application of integrated bioprocess using in situ recovery. With this method, production of human granulocyte-macrophage colony-stimulating factor (hGM-CSF) was investigated in this study. First, optimization of cell concentration during the induction phase for the production of hGM-CSF was examined. As cell concentration increased, the level of hGM-CSF was decreased due to the presence of extracellular proteases. Induction using sugarfree media produced 33% more hGM-CSF. The effects of pH on the binding of hGM-CSF to cationic and anionic exchange resins were also investigated. In terms of stability, optimal pH was found to be 5~7. In the case of using buffer exchange when CM-Sepharose was used as a cationic exchange resin, optimal pH for binding was 4.8 and adsorption yield was 77%. When DEAE-Sepharose was used as an anionic exchange resin, it was 5.5 (74%). Without buffer exchange, optimal pH was 4.6 (84%). From these results, an integrated bioprocess using in situ recovery with simultaneous production and separation of foreign protein in transgenic plant cell suspension cultures was found to be feasible.

• 한국환경성돌연변이발암원학회지
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• 제16권1호
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• pp.13-18
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• 1996

One mechanism of free-radical production by ethanol is suggested to be through the intracellular conversion of XDH to XO by increased ratio of NADH to NAD. The major mechanism for physiological compensation of cytosolic NADH/NAD balance is the malate/aspartate shutfie. Therefore, it is important to develop the method to improve the efficiency of malate/aspartate shuttle in ethanol metabolism. In the present study, various amino acids and organic acid involved in the shuttle were tested for their functional efficiency in modulating shuttle in the ethanol-perfused rat liver. The rate of ethanol oxidation in the liver perfused with aspartate alone or aspartate in combination with pyruvate, respectively, was increased by about 10% compared to control liver, but not in the tissues perfused with glummate, cysteine or pyruvate alone. Though glummate, cysteine and pyravate did not affect the ethanol oxidation significanfiy, they showed some suppresive effect on the ethanol-induced radical generation monitored by protein carbonylation analysis. Among the tested components, aspartate is confirmed to be the most efficient as a metabolic regulator for both ethanol oxidation and ethanol-induced oxidative stress in our perfusion system. These effects of aspartate would result from NAD recycling by its supplementation through the coupled aspartate aminotransferase/malate dehydrogenase reactions and the malate-aspartate shuttle.

• KSBB Journal
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• 제13권1호
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• pp.13-19
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• 1998

Biopolymer membrane was prepared using two oppositely charged natural biopolymers. The biopolymer membrane was used for the encapsulation of two hybridoma cell lines(ATCC CRL-1606, ATCC HB-8852) to produce monoclonal antibodies. In order to reduce the down stream steps, the pre size of the membrane was controlled to retain the monoclonal antibodies in the capsules based on the diffusion experiments with standard proteins. T-flask culture showed cell densities of 8$\times$107 cells/mL and 3$\times$107 cells/mL, and MAb concentrations of 506$\mu$g/mL and 109$\mu$g/mL for encapsulated ATCC CRL-1606 and HB-8852, respectively. Two liter perfusion cultures with encapsulated ATCC HB-8852 were performed to enhance the MAb production. The MAb production of the encapsulated hybridoma increased considerably comparing to the culture using silicon tubing for oxygen transfer.

• Toxicological Research
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• 제2권1호
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• pp.1-7
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• 1986

Procarcinogen induced DNA single-strand breaks and unschduled DNA synthesis were measured in primary rat hepatocytes culture. For DNA single-strand breaks assay, rat liver DNA was prelabeled by injection 3H-thymidine during the peak of DNA synthesis following partial hepatectomy. Hepatocytes were isolated from the rat 2 weeks after surgery by a collagenase perfusion techinique and maintained as monolayers in serum free medium on collagen-coated culture dishes. DNA sigle-strand breaks were measured by the alkaline elution techinique.

• Biotechnology and Bioprocess Engineering:BBE
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• 제5권4호
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• pp.275-287
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• 2000

Fermentation strategies for recombinant protein production in Pichia pastoris have been investigated and are reviewed here. Characteristics of the expression system, such as phenotypes and carbon utilization, are summarized. Recently reported results such as growth model establishment, app58lication of a methanol sensor, optimization of substrate feeding strategy, DOstat controller design, mixed feed technology, and perfusion and continuous culture are discussed in detail.

• Toxicological Research
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• 제3권2호
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• pp.73-80
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• 1987

Primary cultures of adult rat hepatocytes were used to study the cytotoxicity of D-galactosamine. Hepatocytes were isolated by a collagenase perfusion technique and maintained as monolayers in serum-free medium on collagen-coated culture dishes. Treatment of galactosamine to the culture markedly inhibited the uptake of ${\alpha}$-aminoisobutyric acid (AIB) inducible with glucagon and dexamethasone. At0.1 mM of galactosamine, AIB uptake was inhibited significantly when treated for 12 hr. At higher doses (0.25, 0.5 and 1.0mM), a significant inhibition was noticed after 1 hr exposure. Generally the magnitude of the inhibition was related to the dose and treatment time of galactosamine. Treatment of galactosamine also produced a dose- and treatment time-related suppression of the tyrosine aminotransferase (TAT) induction caused by dexamethasone. Meanwhile, uptake of ouabain was not affected by the treatment of galactosamine. The viability of the hepatocytes was decreased only slightly by the treatment of galactosamine; more than 87% of the cells excluded tryphane blue when treated 1 mM galactosamine for 12 hr. Galactosamine induced depressions of AIB uptake and TAT activity were prevented by the simultaneous addition of uridine to the culture. D-Galactosamine, cytotoxicity, hepatocytes culture, ${\alpha}$-aminoisobutyric acid uptake, tyrosine aminotransferase.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2005년도 생물공학의 동향(XVI)
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• pp.37-37
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• 2005

Human chorionic gonadotropin (hCG) is a heterodimeric glycoprotein hormone consisting of non-covalently linked two subunits, the ${\alpha}$ and ${\beta}$ subunit. It has been used as a infertility drug for ovulation to mimic luteinizing hormone $(LH).^{1)}$ A stable cell line was established by transfection of Rc/CMV-i-dhfr-hCG, expression vector containing hCG ${\alpha}-$ and ${\beta}-genes$, into dihydrofolate reductase-deficient CHO cells and subesquent methotrexate-mediated gene amplification. Anchorage-dependent CHO cells were adapted into a serum-free and/or animal component-free suspension medium through gradual serum weaning for the hCG production. The established cell line showed typical morphological characteristics and growth profile of CHO cells, and could produce FSH with passage-to-passage consistency. The high density perfusion culture of the CHO cells was carried out in Celligen Plus bioreactor equipped with a spin-filter as a internal cell retention device. The cell density reached up to $>1x10^{7}$ cells/ml in less than 7 days and a perfusion-control strategy based on cellular consumption rates of glucose was $established.^{2)}$ Biologically active recombinant hCG was purified by a series of chromatographic steps including anion exchange chromatography and hydrophobic interaction chromatography to homogeneity. The highly purified recombinant hCG was characterized for physicochemical, immunological and biological properties.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2000년도 추계학술발표대회 및 bio-venture fair
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• pp.159-162
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• 2000

유가식배양을 통한 고농도배양에서 계면활성제와 산소전달물질의 첨가는 세포의 고밀도와 배지의 고점도로 인해 발생하는 nutrient의 물질전달제한과 산소부족현상을 효과적으로 해결하였다. Pluronic 10R-5는 Pluronic F-68과 같은 triblock copolymer 구조이며 세포생장의 저해를 나타내지 않았다. 그러나 diblock copolymer구조이며 소수성 부분이 hydrocarbon block인 Plurafac A-38은 세포의 생장을 저해함을 관찰할 수 있었다. 따라서 장기간의 유가식배양이나 perfusion 배양을 수행할 경우 세포의 생존도를 증대시켜 유용물질의 생산성을 높일 수 있으며 여러 형태의 bioreactor에서 고농도 배양을 가능케 하여 유용물질 생산성을 높일수 있을 것이라고 기대된다.

• KSBB Journal
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• 제9권2호
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• pp.230-237
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• 1994

회전식 여과기는 일종의 분리기로서 동물세포의 연속배양에 이용되어 높은 세포농도와 그와 비례한 높은 생산성을 가져다 준다. 회전식 여과기를 이용한 세포배양은 여러 인자들에 의해 세포농도의 변화가 결정되는데 이를 수학적으로 modeling하고 수치 모사와 sensitivity analysis를 통하여 조사하였다. 고농도 배양시 암모니아의 축적은 세포 성장을 크게 둔화시키고 최대세포농도도 따라서 낮게 된다. 운전 인자 중 세포유치율은 세포 성장속도와 최대세포농도의 크기에 가장 큰 영향을 끼침이 밝혀졌다. 비배지공급속도는 세포농도의 변화에 거의 영향을 끼치지 않으며 배지의 연속식공급과 계단식공급은 세포성장에 큰 차이를 보이지 않는다.

• Advances in biomechanics and applications
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• 제1권4호
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• pp.227-237
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• 2014

The in vitro construction of osteo-articular large implants combining biomaterials and cells is of great interest since these tissues have limited regeneration capability. But the development of such organoids is particularly challenging, especially in the later time of the culture, when the extracellular matrix has almost filled the initial porous network. The fluid flow needed to efficiently perfuse the sample can then not be achieved using only the hydraulic driving force. In this paper, we investigate the interest of using an electric field to promote mass transport through the scaffold at the late stage of the culture. Based on the resolution of the electrokinetics equations, this study provides an estimation of the necessary electric driving force to reach a sufficient oxygen perfusion through the sample, thus analyzing the feasibility of this concept. The possible consequences of such electric fields on cellular activities are then discussed.