• KSBB Journal
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• v.30 no.3
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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.

• Environmental Mutagens and Carcinogens
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• v.16 no.1
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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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• v.13 no.1
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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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• v.2 no.1
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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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• v.5 no.4
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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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• v.3 no.2
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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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• 2005.04a
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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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• 2000.11a
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• pp.159-162
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• 2000

It has been commonly known that cell growth is inhibited by the lack of dissolved oxygen and mass transfer inhibition of nutrients at stationary phase in fed-batch culture. In this study, Pluronic F-68 and oxygen vectors were added in Angelica gigas Nakai suspension culture in order to enhance cell growth in fed-batch culture. It was observed that the addition of 6%(w/v) Pluronic F-68 promoted cell growth up to 6.1% compared to control and that the use of 4%(v/v) n-hexadecane markedly enhanced cell growth up to 11.4%.

• KSBB Journal
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• v.9 no.2
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• pp.230-237
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• 1994

Spin-filters are used as cell separation devices for achieving high cell density and high productivity in animal cell culture. We have proposed a model for the cell growth in a spin-filter perfusion culture and examined the effects on cell growth by several parameters including ammonia inhibition, specific growth rate, specific feeding rate, and cell retention. Results from computer simulation and sensitivity analysis indicate that the cell retention affects the cell growth mostly while there is a significant inhibition on cell growth by the ammonia accumulated during the culture. The specific feeding rate has minimal effects on cell growth, which is consistant with the fact that the cell growth with a step feeding is quite similar to that with a continuous feeding.

• Advances in biomechanics and applications
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• v.1 no.4
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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.