• BMB Reports
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• 제40권3호
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• pp.439-443
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• 2007

Tumor growth and metastasis are dependent on angiogenesis, and endothelial cell invasion and migration are apparent means of regulating tumor progression. We report here that saxatilin, a snake venom-derived disintegrin, suppresses the angiogenesis-inducing properties of NCI-H460 human lung cancer cells. Culture supernatants of NCI-H460 cells are able to induce human umbilical vascular endothelial cell (HUVEC) invasion and tube formation. However, treatment of the cancer cells with saxatilin resulted in reduced angiogenic activity of the culture supernatant. This suppressed angiogenic property was found to be associated with the level of vascular endothelial growth factor (VEGF) in the culture supernatant. Further experimental evidence indicated that saxatilin inhibits VEGF production in NCI-H460 cells by affecting hypoxia induced factor-1$\alpha$ (HIF-1$\alpha$) expression via the Akt pathway.

• 한국발생생물학회지:발생과생식
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• 제20권2호
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• pp.101-108
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• 2016

Previously we observed that human adipose-derived stem cells (hADSCs) could form aggregation during culture in the presence of human serum (HS). In the present study, we have examined if the aggregation might result from the cell migration and analyzed the difference of cell adhesivity after culture in various conditions. When cells were cultured in fetal bovine serum (FBS) alone, there was no morphological change. Similarly, cells pretreated with FBS for 1 day or cultured in a mixture of FBS and HS showed little change. In contrast, cells cultured in HS alone exhibited formation of cell-free area (spacing) and/or cell aggregation. When cells cultured in FBS or pretreated with FBS were treated with 0.06% trypsin, almost cells remained attached to the dish surfaces. In contrast, when cells cultured in HS alone were examined, most cells detached from the dish by the same treatment. Treatment of cells with forskolin, isobutylmethyl xanthine (IBMX) or LY294002 inhibited the formation of spacing whereas H89 or Y27632 showed little effect. When these cells were treated with 0.06% trypsin after culture, most cells detached from the dishes as cells cultured in HS alone did. However, cells treated with IBMX exhibited weaker adhesivity than HS alone. Based on these observations, it is suggested that HS treatment might decrease the adhesivity and induce three-dimensional migration of hADSCs, in the latter of which cAMP signaling could be involved.

• BMB Reports
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• 제50권11호
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• pp.572-577
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• 2017

In most clinical applications, human mesenchymal stem cells (hMSCs) are expanded in large scale before their administration. Prolonged culture in vitro results in cellular senescence-associated phenotypes, including accumulation of reactive oxygen species (ROS) and decreased cell viabilities. Profiling of stem cell-related genes during in vitro expansion revealed that numerous canonical pathways were significantly changed. To determine the effect of selenocysteine (Sec), a rare amino acid found in several antioxidant enzymes, on the replicative senescence in hMSCs, we treated senescent hMSCs with Sec. Supplementation of Sec in the culture medium in late-passage hMSCs reduced ROS levels and improved the survival of hMSCs. In addition, a subset of key antioxidant genes and Sec-containing selenoproteins showed increased mRNA levels after Sec treatment. Furthermore, ROS metabolism and inflammation pathways were predicted to be downregulated. Taken together, our results suggest that Sec has antioxidant effects on the replicative senescence of hMSCs.

• 한국미생물·생명공학회지
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• 제45권2호
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• pp.93-100
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• 2017

인간노로바이러스는 바이러스성 식중독 원인의 대부분을 차지한다. 노로바이러스가 건강한 성인에 감염하면 설사 등의 병변을 몇 일간 일으키다 대부분 별다른 처치 없이도 치유되는 경우가 대부분이다. 그러나 면역기능이 약화된 환자에게 감염한 경우, 만성감염 내지 치명적 감염도 가능한 것으로 보고 되고 있다. 1968년에 처음 노로바이러스가 보고된 이후 세포 감염모델과 소동물감염 모델을 만들고자 하는 시도가 이어져 왔으나 대부분 실패하였다. 그러나 최근들어 세포감염 모델 개발에 있어 주목할 만한 기념비적인 연구들이 이루어졌다 것이 고무적이다 할 수 있다. 이번 총설에서는 새로 개발된 감염 모델들의 특징과 장단점을 살펴보고, 이를 더욱 개선할 수 있는 방향에 대하여 살펴보고자 한다.

• KSBB Journal
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• 제27권3호
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• pp.186-194
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• 2012

Various human host cell lines, which are more effective than the other original human cell lines, have been developed and used. Highly efficient human cell line can be obtained from the fusion between human embryonic kidney 293 (HEK293) and human Burkitt's lymphoma cells (Namalwa). Fused cell line has the advantages of both cell lines such as the high transfection efficacy of HEK293 cells and the constitutive expression of Epstein-Barr virus (EBV) genome which is related with high expression of target protein and anti-apoptotic growth of Namalwa cells. In this study, characterization of two original cell lines was performed by using design of experiment (DOE) considering cell maintenance, media development, optimization of culture condition, and scale-up. The formation of aggregates was apparent with high glutamine concentration at more than 6 mM. Supplementation of hydrolysates showed positive effects on the growth performances of HEK293 cells. On the contrary, Namalwa cells showed negative results. It was confirmed that Namalwa cells were more sensitive to lower temperature at $35^{\circ}C$ and hyperosmotic condition over 260 mOsm/kg. In addition, both cell lines showed limited growth in 3-L bioreactor due to shear stress.

• Journal of Microbiology and Biotechnology
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• 제10권3호
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• pp.321-326
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• 2000

To examine the feasibility of the long-term production of the human granulocyte colony stimulating factor (hG-CSF) using the $_{L}-arabinose$ promoter system of Escherichia coli, flask relay culture and cyclic fed-batch culture were performed. In the flask relay culture, it was found that the pismid was maintained stably up to about 170 generations in an uninduced condition, whereby the cells could also maintain the capability of expressing hG-CSF expression were maintained stably up to at least 100 generations. In contrast, in the cyclid fed-batch culture, segregational plasmid instability was observed within about 4 generations after induction, even though the cell growth and hG-CSF production reached their maximum balues, 78.0 g/l of dry cell weight and 7.0 g/l of hG-CSF, respectively. It would appear that, when compared to the flask relay culture, the high-cell density and high-level expression of hG-CSF in the cyclic fed-batch cultrure led to the segregational plasmid instability; in other words, a severe metabolic burden existe on the cells due to the high-level expression of hG-CSF. Accordingly, based on these long-term cultures, the segregational and structural plasmid instability was observed and a strategy to overcome such problems could be designed.

• Biotechnology and Bioprocess Engineering:BBE
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• 제11권2호
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• pp.154-159
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• 2006

Interleukin-18 (IL-18), otherwise known as interferon-gamma-inducing factor (IGIF), is one of several well characterized and important cytokines that contribute to host defenses. The complementary DNA (cDNA) of mature human interleukin-18 gene (hIL-18) was fused with the signal peptide of the rice amylase 1A gene (Ramy1A) and introduced into the plant expression vector under the control of a duplicated CaMV 35S promoter. The recombinant plasmid was transformed into tobacco (Nicotiana tabacum L. cv Havana) using the Agrobacterium-mediated transformation method. The integration of the hlL-18 gene into the genome of transgenic tobacco plants was confirmed by polymerase chain reaction (PCR) amplification and its expression was observed in the suspension cells that were derived from the transgenic plant callus by using Northern blot analysis. The hlL-18 protein was detected in the extracts of the transgenic callus and in the medium of the transgenic tobacco suspension culture by using immunoblot analysis. Based upon enzyme-linked immunosorbant assay (ELISA) results, the expression level of the hlL-18 protein approximated $166{\mu}g/L$ in the suspension culture medium. Bioassay results from the induction of $interferon-{\gamma}$ from a KG-1 cell line indicated that the hlL-18 secreted into the suspension culture medium was bioactive.

• Journal of Pharmaceutical Investigation
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• 제34권5호
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• pp.393-399
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• 2004

For the efficient determination of activity against solid tumors, an in vitro tumor model that resembles the condition of in vivo solid tumors, is required. The purpose of this study was to establish a rapid culture method and viability assay for an in vitro 3-dimensional tumor model, multicellular spheroid (MCS). Among 12 human cancer cell lines, a few cell lines including DLD-1 (human colorectal carcinoma cells) formed fully compact MCS which was adequate for in vitro viability assay. DLD-1 MCS showed steady growth reaching $700\;{\mu}m$ diameter after 11 day culture. DLD-1 cells grown as MCS showed significant increase in $G_0/G_1$ phase compared to the monolayer cells (73.9% vs 45.7%), but necrotic regions or apoptotic cells were not observed. The cells cultured as MCS showed resistance to 5-FU (10.3 fold higher $IC_{50}$) compared to monolayers, however, tirapazamine (a hypotoxin) showed similar activity in both culture systems. In summary, MCS may be a valid in vitro model for activity screening of anticancer agents against human solid tumors and also exploitable for studying molecular markers of drug resistance in human solid tumors.

• Clinical and Experimental Reproductive Medicine
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• 제31권4호
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• pp.209-215
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• 2004

Objective: Embryonic stem (ES) cells could be differentiated into the specific cell types by alternation of culture condition and modification of gene expression. This study was performed to evaluate the differentiation protocol for mouse and human ES cells to insulin secreting cells. Methods: Undifferentiated mouse (JH-I) and human (Miz-hESI) ES cells were cultured on STO feeder layer, and embryoid bodies (EBs) were formed by suspension culture. For the differentiation, EBs were cultured by sequential system with three stage protocol. The differentiating ES cells were collected and marker gene expressions were analyzed by seIni-quantitative RT-PCR in each stage. Amount of secreted insulin levels in culture media of human ES cells were measured by human insulin specific RIA kit. Results: During the differentiation process of human ES cells, GATA-4, a-fetoprotein, glucose transporter-2 and Ngn-3 expression were increased whereas OctA was decreased progressively. Insulin and albuInin mRNAs were expressed from stage IT in mouse ES cells and from stage III in human ES cells. We detected 3.0~7.9 IlU/rnl secretion of insulin from differentiated human ES cells by in vitro culture for 36 days. Conclusion: The sequential culture system could induce the differentiation of mouse and human ES cells into insulin secreting cells. This is the fIrst report of differentiation of human ES cells into insulin secreting cells by in vitro culture with serum and insulin free medium.

• Biotechnology and Bioprocess Engineering:BBE
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• 제8권2호
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• pp.135-141
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• 2003

The human granulocyte-macrophage colony stimulating factor (hGM-CSF) gene was introduced into tobacco plants. The cell suspension culture was established from leaf-derived calli of the transgenic tobacco plants in order to express and secrete a biologically active hGM -CSF. The recombinant hGM-CSF from the transgenic plant cell culture (prhGM-CSF) was identified as a yield of about 180 ${\mu}$g/L in the culture filtrate, as determined by ELISA. The addition of 0.5 g/L polyvinylpyrrolidone (PVP) to the plant cell culture medium both stabilized the secreted prhGM-CSF and increased the level of production approximately 1.5-fold to 270 ${\mu}$g/L. The biological activity of the prhGM-CSF was confirmed by measuring the proliferation of the hGM-CSF-dependent cell line, TF-1. Interestingly, the specific activity of the prhGM-CSF was estimated to be approximately 2.7 times higher than that of a commercially available preparation from E. coli.