• 한국발생생물학회지:발생과생식
• /
• 제19권3호
• /
• pp.119-126
• /
• 2015

The suitable feeder cell layer is important for culture of embryonic stem (ES) cells. In this study, we investigated the effect of two kinds of the feeder cell, MEF cells and STO cells, layer to mouse ES (mES) cell culture for maintenance of stemness. We compare the colony formations, alkaline phosphatase (AP) activities, expression of pluripotency marker genes and proteins of D3 cell colonies cultured on MEF feeder cell layer (D3/MEF) or STO cell layers (D3/STO) compared to feeder free condition (D3/-) as a control group. Although there were no differences to colony formations and AP activities, interestingly, the transcripts level of pluripotency marker genes, Pou5f1 and Nanog were highly expressed in D3/MEF (79 and 93) than D3/STO (61and 77) or D3/- (65 and 81). Also, pluripotency marker proteins, NANOG and SOX-2, were more synthesized in D3/MEF ($72.8{\pm}7.69$ and $81.2{\pm}3.56$) than D3/STO ($32.0{\pm}4.30$ and $56.0{\pm}4.90$) or D3/- ($55.0{\pm}4.64$ and $62.0{\pm}6.20$). These results suggest that MEF feeder cell layer is more suitable to mES cell culture.

• Reproductive and Developmental Biology
• /
• 제36권1호
• /
• pp.21-26
• /
• 2012

Suspension culture is a useful tool for culturing embryonic stem (ES) cells in large-scale, but the stability of pluripotency and karyotype has to be maintained $in$ $vitro$ for clinical application. Therefore, we investigated whether the chromosomal abnormality of ES cells was induced in suspension culture or not. The ES cells were cultured in suspension as a form of aggregate with or without mouse embryonic fibroblasts (MEFs), and 0 or 1,000 U/ml leukemia inhibitory factor (LIF) was treated to suspended ES cells. After culturing ES cells in suspension, their karyotype, DNA content, and properties of pluripotency and differentiation were evaluated. As a result, the formation of tetraploid ES cell population was significantly increased in suspension culture in which ES cells were co-cultured with both MEFs and LIF. Tetraploid ES cell population was also generated when ES cells were cultured alone in suspension regardless of the existence of LIF. On the other hand, the formation of tetraploid ES cell population was not detected in LIF-free condition, in which MEFs were included. The origin of tetraploid ES cell population was turned out to be E14 ES cells and not MEFs by microsatellite analysis and the basic properties of them were still maintained despite ploidy-conversion to tetraploidy. Furthermore, we identified the ploidy shift from tetraploidy to near-triploidy as tetraploid ES cells were differentiated spontaneously. From these results, we demonstrated that suspension culture system could induce ploidy-conversion generating tetraploid ES cell population. Moreover, optimization of suspension culture system may make possible mass-production of ES cells.

• Journal of Microbiology and Biotechnology
• /
• 제17권5호
• /
• pp.739-744
• /
• 2007

Insect cell lines and the control of infection for obtaining the maximum amount of polyhedrin-Cry1Ac-polyhedrin fusion protein from Bactrus in monolayer and suspension culture systems were tested. Growth rates of the Trichoplusia ni(High-Five) cell line in both culture systems were better than the other insect cell lines, Spodoptera frugiferda(Sf-9, Sf-21), Trichoplusia ni(Tn5), and Spodoptera exigua(Se301). The expression of the fusion protein in a monolayer culture showed that Se301 cells were 2.3-4.8 times more productive on a per cell basis than the other cell lines. However, in suspension culture, only High-Five cells were productive. High-Five cells infected with Bactrus at a multiplicity of infection(MOI) of 5 and a cell density of $3.0{\times}10^5$ cells per ml were more productive than the other infection condition in a suspension culture suitable for a large-scale production of baculovirus. In conclusion, for the large-scale production of Bactrus in vitro, High-Five cells showing good growth and high productivity are suitable.

• 한국생물공학회:학술대회논문집
• /
• 한국생물공학회 2003년도 생물공학의 동향(XII)
• /
• pp.398-401
• /
• 2003

Bacillus species predominantly outgrown in a night soil treatment system were isolated and characterized. Cell interactions took place among them and cell population changed under various culture conditions. Maximum removal of $NH_4\;^+-N$ and cell production occurred under the conditions of 30% DO and C/N ratio of 8. Additions of 0.8% peptone and 0.3% yeast extract to a basal medium influenced the growth of isolates and the removal of $NH_4\;^+-N$ in flask culture, and metal ions such as Ca, Fe and Mg also did. During the flask experiment of nitrogen removal under an aerobic condition, active nitrification by the isolates occurred largely in 1 h with the decrease of COD and alkalinity destructed was only 74.6% of theoretical value. From the nitrogen balance, the percentage of nitrogen lost in the flask culture was estimated to be 29.0%. This conversion of ammonia to $N_2$ under an aerobic condition was confirmed by GC analysis. The B3 process using the Bacillus species seemed to have some economic advantage.

• 미생물학회지
• /
• 제16권3호
• /
• pp.93-102
• /
• 1978

Saccharomyces cerevisiae strain M was cultured in a molasses-containing media with repeated transplantations of the yeasts from one culture to another to adapt to molasses. After that only different amounts of phosphorous and nitrogen sources were added to the media. And then some variations during the culture time and the effects of consituents of cell mass on the functional activity and sensitivity of the cell were investigated. The results obtained were summarized as follows : 1. In the same culture condition of yeasts, the carbohydrates and trehaloses contents were more remarkably increased when small amounts of phosphate and nitrogen sources were added, then when alrge amounts were added, but yield percentage on assimilated sugars was lower. 2. The content of trehalose in yeast cells was reduced remarkably at the early stage in the culture, but this increased remarkably at later stage. When small amounts of nitrogen and phosphate were added to the culture medium, the amount of thehalose in the cells increased greasly. 3. The more protein content was present in the yeast cells, the smaller the carbohydrate and trehalose content, but more amino-N, RNA and moisture content were present in the cells. And in this case fermentability of the cells was stronger, but sugar tolerance was lower. 4. During the preservation period of compressed yeast cells at different temperature, the higher the temperature was, the more rapidly the amount of trehalose in the cells decreased. And in the cell where the amount of trehalose in the cells decreased. And in the cell where the amount of trehalose(carbohydrate) was large and the amount of protein was small, the amount of trehalose decreased at a slower rate during the preservation period.

• 한국약용작물학회지
• /
• 제12권5호
• /
• pp.366-371
• /
• 2004

Aconitine alkaloids produced from cell suspension cultures of Aconitum napellus for the first time. The effects of various culture conditions on cell biomass and aconitine accumulation in cell suspension cultures were investigated. Suspension cell cultures of A. napellus were established by transferring callus tissues from leaf explants onto liquid MS medium supplemented with $1\;mg/l$ NAA and $0.1\;mg/l$ kinetin. Among the culture media tested, MS medium had a pronounced effect on cell growth and aconitine accumulation. The maximum dry cell weight was obtained at inoculum size of 3 g (FCW) per flask and in MS medium supplemented with 5% sucrose after 8 weeks. The addition of salicylic acid (SA) and yeast extract (YE) in the MS medium enhanced aconitine accumulation. Using a proper combination of culture condition and supplements, aconitine content could reach 0.043% (dry weight basis), that was $2.5{\sim}3$ fold higher that detected in control cultures.

• Toxicological Research
• /
• 제21권1호
• /
• pp.57-62
• /
• 2005

To investigate the mutant induction of wild ginseng culture extract, we performed chromosomal aberration assay with chinese hamster lung cell in vitro. The test concentration of the extract was decided for the standard with the 50% suppression of cell propagation in the cell. The concentrations for the chromosome test were 1,250, 2,500 and 5,000 ㎍/ml with metabolic activation (+S, 6 hours treatment), 1,100, 2,200 and 4,400 ㎍/ml without metabolic activation (-S, 6 hours treatment) 800, 1,600 and 3,200 ㎍/ml without metabolic activation (-S, 24 hours treatment). No significant increase in chromosome aberrations was observed at any of these concentrations both in the absence and presence of metabolic activation system. Cyclophosphamide monohydrate (CPA) and ethylmethanesulfonate (EMS) caused a significant increase in chromosome aberration. These results may be concluded that wild ginseng culture extract is not capable of inducing chromosome aberration in cultured chinese hamster lung cell regardless of metabolic activation and genotoxicity of that is negative under the present experimental condition.

• 대한의용생체공학회:의공학회지
• /
• 제32권3호
• /
• pp.185-190
• /
• 2011

The purpose of this study is to investigate the influence of organism vigor information solution(OVIS) on the animal cell growth and to set up a condition for cell culture. We investigated the reactions on the MG-63 and MCF-7 cell line in mixture culture media with various amount of REVIEW solution, which is an example among various OVIS. DMEM-HG was used as basic media. The concentration range of the mixture was limited from 0% to 15%. MTT assay is used for cell viability test. The cell was incubated for 14days and the MTT assay was performed on day 1, 3, 7, 10 and 14 throughout the experiment. We used the ELISA reader to measure the Optical Density(O.D) at 595 nm wavelength filter. MCF-7 was linearly proliferated according to culture time and concentrations of OVIS. On the other hand, MG-63 cells were measured the highest O.D value at 12%. The growth rates of both cells cultured in mixed culture media with OVIS are much higher than those in only basic media, DMEMHG, after 14days. It was confirmed that cell cultured at OVIS grows rapidly at certain period although cells showed a negative effect in initial stage.

• Clinical and Experimental Reproductive Medicine
• /
• 제30권1호
• /
• pp.65-75
• /
• 2003

Objectives: To investigate the role of TGF (Transforming growth factor-$\beta$) involved in the paracrinic communication during decidualization between UEC (uterine epithelial cells) and USC (uterine stromal cells), we have employed a co-culture system composed of human endometrial epithelial and stromal cells in defined hormonal conditions. Design: In the co-culture, endometrial epithelial cells cultured in the matrigel-coated cell culture insert are seeded on top of the endometrial stromal cells cultured within a collagen gel. The co-culture was maintained for 48 hours under the following hormonal conditions: progesterone dominant condition (100 nM P4 and 1 nM E2) or estrogen-dominant condition (100 nM E2 and 1 nM P4). 10 ng/ ml HGF and/or 10 ng/ml TGF-$\beta$1 are added. Methods: RT-PCR is utilized to detect mRNAs quantitatively. Enzyme-linked immunosorbent assay (ELISA) and immunohistochemical staining are utilized to detect proteins in the tissue. Results: Prolactin mRNA is expressed in the co-cultured stromal cells under the progesterone dominant condition. TGF-$\beta$1 and its receptors are expressed in both the co-cultured epithelial and stromal cells irrespective of the steroid present, which is in contrast with no or negligible expression of TGF-$\beta$1 or its receptor in cells separately cultured. Both estrogen and progesterone significantly elevate the concentration of hepatocyte growth factor (HGF) in the conditioned medium of the co-culture with the value of 4, 325 pg/ml in E2-dominant and 2, 000 pg/ml in P4-dominant condition compare to 150 pg/ml in no hormone. In separately cultured stromal cells, administration of HGF induces the expression of TGF receptor 1 in both hormonal conditions, but induction of TGF receptor 2 is only manifest in the P4-dominant condition. Administration of TGF-$\beta$ and HGF directly induce the decidualization marker prolactin mRNA in separately cultured stromal cells. Conclusion: It is likely that steroid hormones induces prolactin mRNA indirectly by promoting the cell to cell communication between the stromal and the epithelial cells. TGF-$\beta$ and HGF are two possible paracrine mediators in the human endometrial decidualization.

• 한국생물공학회:학술대회논문집
• /
• 한국생물공학회 2003년도 생물공학의 동향(XIII)
• /
• pp.273-277
• /
• 2003

Batch culture of mutant derived from Pichia ciferrii ATCC 14091 was investigated for producing the intracellular tetraacetylphytosphingosine (TAPS). Composite experimental design was used to optimize the composition of the culture medium for maximizing the productivity of TAPS. In this experiment, various culture parameters were investigated that were the effects of temperature, the initial culture pH, the carbon-to-nitrogen ratio, the concentration of trace elements, and the concentration of cofactors. The optimal temperature for cell growth and TAPS synthesis appeared to be %25^{\circ}C$. An initial pH value of 7.5 gave the best results. Under the best condition, the maximum TAPS concentration indicated 7.2 g/L and its productivity was 0.06 g/L-hr in a 2.5 L jar