• Reproductive and Developmental Biology
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• v.38 no.4
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• pp.143-146
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• 2014

Antioxidants, as reactive oxygen species scavengers, are one of the beneficial additives in serum-free defined culture medium. In this study, three separate experiments were performed to determine the effects of 3-hyroxyflavone added to the culture medium on the developmental competence of follicular bovine oocytes during in vitro maturation (IVM) and/or in vitro culture (IVC). The rate of blastocyst developed from oocytes cultured in IVM medium with 3-hyroxyflavone was significantly higher than that from control oocytes (39.0% vs. 26.3%, p<0.001), respectively. However, oocytes cultured in the medium with addition of 3-hyroxyflavone only at IVC period did not show significance in the blastocyst development when compared with control. When 3-hyroxyflavone was added to both IVM and IVC media, the rate of blastocyst formation was even significantly lower (21.1%) than control (26.5%; p<0.05). The present findings suggested that antioxidative activity of 3-hydroxyflavone added to only IVM medium beneficially affected the developmental competence of follicular bovine.

• Clinical and Experimental Reproductive Medicine
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• v.20 no.2
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• pp.165-176
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• 1993

These studies were carried out to investigate the optimal freezing protocol for 2 cell mouse embryos and to find the probability of quality control with 2-cell embryos frozen. The embryos showed the best survival by the protocol composed of a freezing solution with the cryoprotectants(1.5M propanediol + 0.1M sucrose), and a 2-steop thawing method(room temperature, 20 sec-37$^{\circ}C$, 20 sec). The developmental ability of frozen-thaw 2-cell embryos did not differ from that of fresh 2-cell embryos in m-KRB medium with 0.4% bovine serum albumin. But development of frozen-thaw embryos was depended on the supplements of the medium. In the albumin-free medium, the developmental rate(rate of blastocysts) was significantly reduced, compared with that in the medium with 0.4% BSA. Also, when frozen-thaw embryos were cultured in the meduim with human fetal cord serum(HCS), the developmental rate of frozen-thaw embryos was sligtly reduced, compared with that of fresh 2-cell embryos. Finally, frozen-thaw 2-cell mouse embryos were more sensitive to the toxic agent of disposable-plastic syringe. Therefore, toxicity of medium could be effectively detected by frozen-thaw 2-cell mouse embryos.

• Journal of Embryo Transfer
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• v.15 no.3
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• pp.231-236
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• 2000

This study was conducted to examine the effects of exogenous gonadotropins (PMSG+hCG) and an antioxidant (cysteine) on in vitro maturation of bovine follicular oocytes. Cumulus-oocyte complexes (COCs) aspirated from 2 to 5 mm ovarian follicles were cultured for 22 to 24 hours in a modified bovine embryo culture medium (mBECM) supplemented with 3 mg/mL bovine serum albumin, to which PMSG (10 IU/mL) + hCG (10 IU/mL) and/or cysteine (0.6 mM) were added. When examined the expansion of cumulus ce1ls at the end of maturation culture, greater (p<0.05) expansion was found after addition of PMSG+hCG (79 to 96%) to mBECM than after no addition (0%), regardless of the presence or absence of cysteine in the medium. The addition of cysteine did not stimulate cumulus expansion, but a high proportion (92%) of expansion was achieved when COCs were cultured after the addition of PMSG+hCG and cysteine to the medium. No difference in the proportion of oocytes underwent germinal vesicle breakdown (initiation of maturation) was found after the addition of PMSG+hCG and/or cysteine to mBECM. However, nuclear maturation (development to the metaphase-II stage) of oocytes was significantly stimulated by the combined addition of PMSG+hCG and cysteine, compared with no addition. In conclusion, both exogenous gonadotropins and an antioxidant are important for nuclear maturation of bovine immature oocytes and these factors have a cell-specific stimulatory action.

• Korean Chemical Engineering Research
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• v.44 no.1
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• pp.73-80
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• 2006

This study compared cell expansion and colony forming ability in human cord blood stem cells cultured ex vivo with two kinds of cytokine combinations, two kinds of media, presence or absence of fetal bovine serum (FBS) and two or three dimensional (2D or 3D) culture environments. Purified $CD34^+$ cells were cultured in the IMDM (Iscove's Modified Dulbecco's Medium) and SFM (Serum Free Medium) containing a cytokine cocktail-I (coc-I) (EPO, GMCSF, SCF, and IL-3) or a cytokine cocktail-II (coc-II) (TPO, G-CSF, SCF, IL-6, and Flt3/Flk-2 ligand) with or without FBS. Generally, higher cellular and clonogenic expansion were observed in the coc-I cytokine condition, compared to coc-II cytokine condition. 3D (Methocult) and 2D (IMDM + coc-I + FBS) conditions gave the greatest cell ($2,258{\pm}456$ fold) and CFU (BFU-E: $652{\pm}19$, CFU-GM: $520{\pm}58$, CFU-GEMM: $339{\pm}100$ fold) expansions, respectively. In aspect of medium, IMDM was better than SFM, except for coc-II condition without FBS. In conclusion, 'IMDM + coc-I + FBS' and 'IMDM + coc-I' were the best CFU expansions on the occasion of all culture conditions. FBS and 2D conditions had affirmative effect on CFU expansion, generally. These data might provide a variety of notions about ex vivo expansion of hematopoietic stem cells.

• Microbiology and Biotechnology Letters
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• v.22 no.4
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• pp.360-367
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• 1994

The effect of various nutrients for the production of the anti-complementary polysaccha- ride by Flammulina velutipes, was examined and the partial purification of the polysaccharide was carried out. The culture conditions and medium compositions considerably influenced the anti-complementary activity of the polysaccharides. When a culture was carried out at 23$\circ$C and pH 5.5 for 6 days in the synthetic medium supplemented with galactose and NaNO$_{3}$, the production of the anti-complementary polysaccharide was maximized. The crude polysaccharide, FV-1 was isolated from the culture broth and partially purified into two fractions, FV-1-II and FV-1-III by gel filtration using Sephadex G-100. FV-1-II was mainly consisted of xylose, mannose, galactose and glucose, and rhamnose, mannose and galactose, for FV-1-III. Also, the anti-complementary activity of FV-1-III was reduced partially in the absence of the Ca ion. When crossed immunoelect- rophoresis using anti-human C3 serum was carried out after incubation of normal human serum with the FV-1-III in the Ca ion free condition, a cleavage of C3 precipitin line was observed. These results indicate that the mode of complement activation by polysaccharide from Flammulina velutipes is via not only the classical pathway but also the alternative pathway.

• Journal of the Korean Society of Food Science and Nutrition
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• v.28 no.3
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• pp.677-684
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• 1999

This study has dealt the effect of Ca regulating hormones and dietary Ca levels on Ca metabolism. Animals(BALB/c mice) were divided into three dietary groups(high and medium Ca and Ca free) and hormones including parathyroid hormone(PTH), calcitonin(CT), cholecalciferol(Vit D) were i.p. injected. After feeding experimental diets for five weeks, mice were anaethetized and sacrificed by heart puncture. We found that femur growth of mouse was slightly increased by high dietary Ca without showing statistical significance comparing to low dietary Ca group. The combination of PTH and CT showed the same effect when dietary Ca was high. At the same time, total mineral retention in bone was most affected by dietary Ca. In general, high Ca diet elevated Ca level in the serum. When dietary Ca was low, PTH stimulated Ca release from the bone into the serum, which was shown to be inhibited by CT treatment. Comparing to the control, PTH, Vit D and CT together tended to inhibit serum Ca level at high and medium dietary Ca. PTH and Vit D inhibited Ca reserve in the liver at all dietary levels of Ca. Both PTH and Vit D stimulated bone Ca retention when dietary Ca was low, but this effect was reversed when dietary Ca was high. When PTH, Vit D and CT were administered together, bone Ca level was greatly enhanced at low dietary Ca than at high dietary Ca, which suggests that these hormonal cooperation is needed for proper bone density maintenance especially when dietary minerals are not sufficient.

• Archives of Pharmacal Research
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• v.28 no.2
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• pp.220-226
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• 2005

Temperature and medium composition were changed with the aim of increasing growth and erythropoietin (EPO) production in EPO-producing Chinese hamster ovary (CHO) cells. We used the CHO cell line, IBE, and its derivative, CO5, which over-expresses the first two enzymes of the urea cycle, carbamoyl phosphate synthetase I (CPS I) and ornithine transcar-bamoylase (OTC). When supplements were added to the medium at $33\;^{\circ}C$, the growth of IBE and CO5 cells increased by $27\%\;and;26\%$, respectively and the maximum yield of EPO was increased by $40\%$ in both cell lines. The absolute EPO concentration in the CO5 cells was always $55{\sim}60\%$ higher than in the IBE cells. In addition, when the two cell lines were continuously cultured with supplements at $33\;^{\circ}C$ until their growth rates approached those at $37\;^{\circ}C$, the growth rates of both IBE and CO5 cells increased by $54\%$ and their maximum EPO levels increased by up to $73\%\;and\;56\%$, respectively. Therefore, the growth and EPO expression levels of CO5 cells increased 2.2-fold and 2.6-fold, respectively, compared to those of the IBE cells. These results indicate that adaptation to lower temperature as well as medium supplementation could be important for improving cell growth and EPO production.

• Korean Chemical Engineering Research
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• v.44 no.1
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• pp.58-64
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• 2006

We successfully isolated human anagen hair follicles from human scalp skin by microdissection and tried to culture them under various conditions. First we confirmed negative effect of serum on human hair follicle organ culture. As a next step serum-free medium compositions, Philpott medium, IMDM, and DHGM (Dongguk hair growth medium) were tried. Philpott medium is a general medium for hair organ culture based on Williams' E medium and DHGM is a special self-developed medium containing high amino acids and vitamins (B group) composition. As results, hair follicle in Philpott medium and IMDM showed anagen phase morphological structure, but rapid loss of hair elongation, low alkaline phosphatase expression, and very low expression of CK19. It is thought these hair follicles rapidly regressed from apoptosis. However, hair follicles in DHGM showed long term anagen phase morphological structure, continuous hair elongation, high alkaline phosphatase, and CK19 expression. These results demonstrate that high amino acids and vitamins (B group) composition are essential to in vitro long term human hair follicle organ culture and this culture medium will be useful in basic study of hair biology or application study to the development of alopecia treatment drugs.

• Korean Journal of Animal Reproduction
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• v.14 no.3
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• pp.183-190
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• 1990

These studies were carried out ot investigate the effects of estrous cow serum(ECS), fetal calf serum(FCS), bovine follicular fluid(BFF) and matured cumulus cell(MCC) on in vitro maturation and fertilization of bovine follicular oocytes. The ovaries were obtained from slaughtered Korean native cows. The follicular oocytes surrounded with cumulus cells were recovered by aspirating follicular fluid from the visible follicles of diameter 3-5mm. The follicular oocytes were cultured in TCM-199 medium containing hormones, FCS, ECS, BFF and MCC for 24~48 hrs. in a incubator with 5% CO2 in air at 38.5$^{\circ}C$ and then matured oocytes were again cultured for 18$^{\circ}C$20 hrs. with motile capacitated sperm in the TCF(Tyrode calcium-free) solution containing 200$\mu\textrm{g}$/ml of heparin. The results obtained in these experiments were summarized as follows : 1. The oocytes classified as "A, B, C, D and Degenerative" depending morphological integrity and those were 61.4%, 12.1%, 19.2%, 4.2% and 3.0% of the total oocytes harvested, respectively. The maturation and fertilization rate of the A, B, C class follicular oocytes, cultured in the TCM-199 medium supplemented with 10% FCS were 89.1%, 78.0%, 52.6% and 78.1%, 33.3%, respectively. 2. The maturation and fertilization rate of the follicular oocytes cultured in TCM-199 medium supplemented with 5%~20% ECS and FCS were 74.0%~80.6, 26.2%~30.0% and 71.7%~76.9%, 51.9%~58.0%, and those values were higher the supplement of ECS than FCS. 3. The maturation rate(68.0%~64.6%) and fertilization rate(59.6%~60.4%) of follicular oocytes cultured in TCM-199 medium supplemented with 10% FCS and 20~30% BFF were higher than those of follicular oocytes cultured TCM-199 medium supplemented with 10% FCS and 10% and 50% BFF. 4. The maturation rate(76.5%) and fertilization rate(61.7%) of follicular oocytes cultured in TCM-199 medium supplemented with 10% FCS and 1$\times$106/ml cumulus cells were higher than those of follicular oocytes cultured in TCM-199 medium supplemented with 10% FCS and 1$\times$104~5/ml and 1$\times$108/ml cumulus cells.lus cells.

• Applied Biological Chemistry
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• v.42 no.3
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• pp.229-234
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• 1999

The effects of linoleic acid(LA, 18 : 2) and/or bovine serum albumin(BSA) on the lipid metabolism in human hepatoma cell line HepG2 cells were evaluated. HepG2 cells were cultured in basal Dulbecco's modified Eagle's(DME) medium(Basal medium), DME medium containing 0.2 mM LA(LA medium), or DME medium containing both 0.2 mM LA and 0.2-1.0% BSA(LA+BSA medium). $[^{14}C]Acetate(0.3\;{\mu}Ci/ml\;medium)$ was added as a radioactive lipid precursor and the cells were incubated for 6 hours. An addition of LA to basal medium resulted in a decrease in the incorporation of $[^{14}C]acetate$ into total cholesterol fraction. In contrast, an addition of BSA to LA-containing medium tended to increase the incorporation of $[^{14}C]acetate$ into total cholesterol. The alteration of cholesterol metabolism in HepG2 cells incubated in LA+BSA medium was attributed by an increase in the incorporation of $[^{14}C]acetate$ into free cholesterol, but not cholesteryl ester fraction. In addition, the secretion of cholesterol was increased by LA+BSA medium, suggesting that BSA stimulates cholesterol secretion. No significant change in the incorporation of $[^{14}C]acetate$ into cellular total lipids was observed among the experimental groups. However, an increased incorporation of $[^{14}C]-labelled$ fatty acid into cellular triacylglycerol and decreased incorporation into phospholipid were observed in cells incubated with LA+BSA medium as compared to those of LA medium. The secretions of $[^{14}C]-labelled$ triacylglycerol, phospholipid, and free fatty acid were also stimulated in HepG2 cells incubated with LA+BSA medium. In conclusion, the present study suggests that in human hepatocytes, LA and BSA influence lipid metabolism, and BSA enhances the secretion of lipids.