• KSBB Journal
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• v.31 no.4
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• pp.284-290
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• 2016

In this research, recombinant human cytotoxic T-lymphocyte antigen 4-immunoglobulin (hCTLA4Ig) was produced by transgenic rice cells. RAmy3D promoter was used for overcome the limitation of low expression level in transgenic plant cells, and the secretion of target protein was accomplished by signal peptide. However, the RAmy3D promoter system which can be induced only by sugar starvation causes the decrease of cell viability. As a result, cell death promotes the release of protease which degrades the target proteins. The protein stability and productivity can be significantly influenced by proteolysis activity. Therefore, development of new strategies are necessary for the in situ recovery of target proteins from cell culture media. In this study, in situ recovery was performed by various strategies. Direct addition of Protein A resin with nylon bag leads to cell death by increased shear stress and decrease in production of hCTLA4Ig by protease. Medium exchange through modified flask could recover hCTLA4Ig with high cell viability and low protease activity, on the other hand, the productivity was lower than that of control. When in situ recovery was conducted at day 7 after induction in air-lift bioreactor, 1.94-fold of hCTLA4Ig could be recovered compared to control culture without in situ recovery. Consequently, in situ recovery of hCTLA4Ig from transgenic rice cell culture could enhance productivity significantly and prevent degradation of target proteins effectively.

• Journal of Applied Biological Chemistry
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• v.43 no.3
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• pp.176-179
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• 2000

Nonpolar taxoides extracted from a large-scale cell culture of Taxus chinensis were isolated through the normal and reverse phase column chromatographies, and their compounds were identified via NMR spectroscopy. The complete separation method was systematically established and described. In dichloromethane, dissolved paclitaxel and other taxoids with hexane were precipitated during the purification of paclitaxel from the plant cell culture of T. chinensis through a large-scale process while the relatively nonpolar taxane derivatives remained dissolved in the hexane phase. 13-Deoxy baccatin III (I), baccatin VI (II), taxchinin I (III), $2{\alpha}$, $5{\alpha}$, $10{\beta}$, $14{\beta}$-tetraacetoxy-4(20), 11-taxadiene(IV), 1-deoxy baccatinVI(V), and taxayuntin C (VI) were isolated through column chromatography and identified via NMR spectroscopy. Compounds I and IV were found to the major components, aside from paclitaxel, in the plant cell culture of T. chinensis. The concentrations of I and IV were compared with the that concentration of the paclitaxel in each of plant cell culture. The possible applications of compounds I, II, IV, and V were discussed.

• Natural Product Sciences
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• v.11 no.3
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• pp.179-182
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• 2005

We investigated that water extract of Acanthopanax sessiliflorus roots rescued the N-methyl-D-aspartate (NMDA), agonist of glutamate receptor, -induced toxicity in rat organotypic hippocampal slice culture. When the cell death in NMDA only-treated hippocampal slices was set 100%, A. sessiliflorus decreased the cell death to 75.4, 51.6, 48.9, and 40.6% at 1, 10, 50, and $100\;{\mu}g/ml$ treatment, respectively. On the basis of these results, the water extract of A. sessiliflorus roots may be a preventive agent against NMDA-induced cytotoxicity.

• Journal of Plant Biology
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• v.36 no.3
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• pp.211-218
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• 1993

Plant regeneration was accomplished from protoplast culture of rice (Oryza sativa L. cv. Taebaeg). Embryogenic callus was induced from mature seed on MS medium containing 5 mM proline, 2.5 mg/L 2,4-D, 30 g/L sucrose in the dark at 28$^{\circ}C$ and used to establish embryogenic cell suspension culture. Suspension cells were subcultured every one week in N6 medium supplemented with 5 mM proline, 200 mg/L casein hydrolysate, 2.5 mg/L 2,4-D and amino acids of AA medium. Suspension cultures were composed of cells that were densely cytoplasmic, potentially embryogenic and were at least maintained for more than 6 months in liquid medium. Protoplasts were isolated from fast-growing suspension culture cells and cultured in a slightly modified KpR medium by mixed nurse culture. Isolated protoplasts began to divide within 5~7 days and thereafter, protoplast-derived calli were sequentially transferred to callus proliferating medium that soft agar MS medium contained 2 mg/L 2,4-D and produced distinct embryogenic cells. Microcolonies were then transferred to solid medium which consisted of MS medium containing 5 mg/L kinetin, 1 mg/L NAA, 1 mg/L ABA, 30 g/L sucrose and 10 g/L sorbitol under fluorescent light. Mulitple shoots of 4~5 per callus emerged and were transferred to hormone-free MS medium for root initiation. Thereafter, The plantlets were transferred to pots of soil to mature in the culture room.

• Journal of the Korean Society of Food Science and Nutrition
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• v.27 no.1
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• pp.141-148
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• 1998

This study describes the effects of 1,25-dihydroxyvitamin D3[1,25(OH)2D3, calcitriol] analog, 1,25(OH)2-16ene-23yne-D3 on proliferatin and differentiatin of the human histiocytic lymphoma cell line U937. This paper also describes the effects of 1,25(OH)2-16ene-23yne-D3 on ${\gamma}$-interferon(IFN-${\gamma}$) synthesis by phytohemagglutinin-activated peripheral blood lymphocytes(PBLs). In the present investigation, 1,25(OH2)-16ene-23yne-D3 was compared to the natural metablite of vitamin D3, 1,25(OH)2D3. 1,25(OH)2-16ene-23yne-D3 was more potent than 1,25(OH)2D3 for inhibition of proliferation and induction of differentiation of U937 cells, Its effects on inhibition of proliferation was about 30-fold more potent than 1,25(OH)2D3. On induction of differentiation as measured by nonspecific esterase (NSE) activity and morphologic change, this analog morphologically and functionally differentiated U937 cells to monocyte-macrophage phenotype showing a decrease of N/C ration in Giemsa staining and the increase of adherence ability of surface. After 3 days in culture, a more significant supression of IFN-${\gamma}$ synthesis analog on supression of IFN-${\gamma}$ synthesis was a dose-dependent manner, with peak activity at 10-7M. The strong direct effects of 1,25(OH)2-16ene-23yne-D3 on cell proliferation and cell differentiation, make this compound an interesting candidate for clinical studies for several types of malignancies, and the effects on supression of IFN-${\gamma}$ synthesis provide the further evidence for a role of 1,25(OH)2-16ene-23yne-D3 in immunoregulation.

• Journal of the Optical Society of Korea
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• v.16 no.1
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• pp.42-46
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• 2012

We have developed a fluorescence optical detection system using a digital micromirror device (DMD) for monitoring 3D cell culture matrices in situ. Full 3D imaging with fast scanning speed was implemented by the combined action of a DMD and a motorized stage. Imaging results with fluorescent microbeads measure the minimum axial resolution of the system as $6.3{\mu}m$, while full 1-mm scanning through 3D alginate-based matrix was demonstrated. For cell imaging, improved images were obtained by removing background fluorescence although the scanning distance was reduced because of low intracellular fluorescence efficiency. The system is expected to be useful to study various dynamics and behaviors of 3-dimensionally cultured cells in microfluidic systems.

• KSBB Journal
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• v.10 no.2
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• pp.191-195
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• 1995

Using Pseudomonas sp. CH-414, the optimum culture conditions were investigated for the cell growth and the phospholipid production in batch culture by varying pH and aeration rate. With starting the cultivation under the conditions of pH 7.0 and 1vvm, pH was controlled to 6 or 8 at 30 hours of culture time. In the case of changing into pH 6.0, the phospholipid production was increased by ca. 20% with comparison to the case of pH 7.0. However, the biomass and the phospholipld concentration were rapidly decreased after 30 hours of culture time when pH was controlled to 8.0. As the aeration rate was increased, the biomass was increased while the phospholipid concentration was considerably varied and unstable. Especially, the concentration of phospholipid was rapidly decreased with 3vvm of aeration rate. Finally, under the culture conditions of pH 7.0 and 3vvm until 30 hours for the cell growth, which were controlled to pH 6.0 and 1vvm for the stable production of phospholipid beyond that time, the dry cell weight was $18.5g/\ell$ and the phospholipid concentration was $\0.83g/ell$ (45mg/g cell).

• Journal of Embryo Transfer
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• v.31 no.1
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• pp.19-25
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• 2016

This study was conducted to evaluate the effects of type of culture media (BM, G2, OS, TCM, and MEM) on B6D2F1 mice oogenesis. In the present study, B6D2F1/CrljOri $F_1$ mice were utilized in order to maximize oogenesis. Also we used TCM-199, Dulbecco's medified Eagle's medium (DMEM), embryo culture medium (Fertilization medium, Cleavage medium, Blastocyst medium), G series medium and One step medium. In vitro maturation was highest in BM followed by the order of OS, MEM, TCM and G2 ($90{\pm}2.8%>88{\pm}3.2%>85{\pm}4.9%>78{\pm}10.2%>64{\pm}7.7%$, respectively). To note, the G2 group was statistically different compared to other groups (p<0.05). On the other hand the fertilization rate was highest in the G2 group followed by BM, OS, TCM, and MEM ($87{\pm}7.2%>85{\pm}6.9%>74{\pm}14.0%>71{\pm}13.8%>2{\pm}1.4%$, respectively). The MEM group was significantly lower compared to other groups (p<0.05). The developmental rate was highest in the OS group followed by the G2 group and the BM group albeit no statistical significance was noted ($73{\pm}11.6%>71{\pm}9.2%>66{\pm}10.4%$). Of note, all cells of the TCM and MEM groups were died during embryonic development. The zona hatched rate ($51{\pm}9.8%$ vs. $50{\pm}9.1%$ vs. $47{\pm}7.2%$ for BM, G2, and OS respectively) and attached rate ($45{\pm}12.3%$ vs. $38{\pm}16.1%$ vs. $37{\pm}11.5%$ for BM, G2, and OS respectively) were not different amongst groups. No difference was found in total cell numbers ($74{\pm}13.9$ vs. $64{\pm}9.2$ vs. $76{\pm}6.7$ for BM, G2, and OS respectively), ICM cell numbers ($20{\pm}1.9$ vs. $14{\pm}1.8$ vs. $15{\pm}2.1$), TE cell numbers ($55{\pm}12.5$ vs. $49{\pm}10.7$ vs. $61{\pm}5.9$), % ICM ($30{\pm}2.8%$ vs. $24{\pm}7.0%$ vs. $22.8{\pm}2.2%$) and ICM:TE ratio ($1:2{\pm}0.5$ vs. $1:3.1{\pm}0.8$ vs. $1:3.1{\pm}0.5$) amongst groups. In summary, these results can provide fundamental data to maximize culture condition for in vitro fertilization on B6D2F1 mice.

• Proceedings of the Korean Vacuum Society Conference
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• 2013.08a
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• pp.277.2-277.2
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• 2013

Petri dishes and glass slides have been widely used as general substrates for in vitro mammalian cell cultures due to their culture viability, optical transparency, experimental convenience, and relatively low cost. Despite the aforementioned benefit, however, the flat two-dimensional substrates exhibit limited capability in terms of realistically mimicking cellular polarization, intercellular interaction, and differentiation in the non-physiological culture environment. Here, we report a protocol of culturing embryonic rat hippocampal neurons on the electro-spun polymeric network and the results from examination of neuronal cell behavior and network formation on this culture platform. A combinatorial method of laser-scanning confocal fluorescence microscopy and live-cell imaging technique was employed to track axonal outgrowth and synaptic connectivity of the neuronal cells deposited on this model culture environment. The present microfiber-based scaffold supports the prolonged viability of three-dimensionally-formed neuronal networks and their microscopic geometric parameters (i.e., microfiber diameter) strongly influence the axonal outgrowth and synaptic connection pattern. These results implies that electro-spun fiber scaffolds with fine control over surface chemistry and nano/microscopic geometry may be used as an economic and general platform for three-dimensional mammalian culture systems, particularly, neuronal lineage and other network forming cell lines.

• Proceedings of the SCSK Conference
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• 2003.09a
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• pp.700-718
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• 2003

Fructan, a polysaccharide existing in plants or produced by microorganisms, is a sugar polymer of fructose with $\beta$-2,6 linkages. In this study, we investigated some cosmeceutical properties of Fructan such as moisturizing effect, cell proliferation effect, anti-inflammation effect and cell cytotoxicity. Zymomonas mobilis, a microorganism producing Fructan, was cultured in a medium containing 10％ sucrose and 2％ yeast extract as main components for 24 hours at 37$^{\circ}C$ and pH 7. Fructan was obtained by precipitation from the cultured medium by adding alcohol (alcohol ratio of 1:3) after removing the enzyme by centrifuging. Fructan exhibited almost same moisturizing effect as hyaluronic acid and cell proliferation effect on human fibroblast and keratinocyte as well. Moreover, on cell proliferation test on bio-artificial skin constructed by 3-dimensional(3-D) culture after inducing primary skin inflammation with 0.5％ sodium lauryl sulfate (SLS), the 3-D artificial skin treated with 0.01 mg/ml, 0.05mg/ml of Fructan exhibited higher cell proliferation than the 3-D artificial skin treated with SLS only. On anti-inflammation test on 3-D artificial skin evaluated by measuring secreted quantity of interleukin-1$\alpha$ (IL-1$\alpha$) which is a pre-inflammatory mediator induced by SLS, the quantity of IL-1$\alpha$on the 3-D artificial skin treated with 0.01 mg/ml, 0.05mg/ml of Fructan was less than the one on the 3-D artificial skin treated with SLS only. As a result of these studies, Fructan has anti-inflammation effect against inflammatory reaction by a skin irritant as well as cell proliferation effect in bio-artificial skin. Fructan was also evaluated as a safe material without any toxicity in safety tests using fibroblasts and animals.