• Korean Journal of Animal Reproduction
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• v.17 no.2
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• pp.113-120
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• 1993

To provide the optimal culture conditions for the developm,ent of in vit개 produced embryos, we have been investigated various culture media as well as co-cultrue systems using porcine cumulus cells or mouse fetal fibroblast cells. Porcine ovaries were brought to the laboratory from local slaughter house within 1 hour after slaughtering and cumulus oocytes complexes were recovered from antral follicles(3~5mm) with 23 gauge needle. To maturate follicular oocytes, cumulus oocytes complexes were washed three times with TCM-199 containing 25mM HEPES and incubated(39$^{\circ}C$, 5% CO2 in air) in various maturation media for 42 hrs. Ejaculated and liquid storaged boar spermatozoa capacitated with different sperm capacitation methods and media were rpepared for fertilizing of matured follicular oocytes in vitro. Fertilization was performed by adding 5~10${\mu}\ell$ fo capacitated spermatozoa containing 1~5$\times$105 sperm/ml to droplets. Eighteen to twenty-eight hours after sperm insemination, fertilized eggs were washed three times with culture media and transferred to the various culture media, to the culture media with a monolayer of somatic cells. The in vitro development rates of 1-cell embryos cultured with three times with culture media and transferred to the various culture media, to the culture media with a monolayer of somatic cells. The in vitro development rates of 1-cell embryos cultured with three different media, m-KRB, BECM and TCM-HEPES were 0~1.0%, showing extremely lower rates. Especially, most of embryos were observed to arrest the development beyond 4-cell stages. The rates of embryos developed to 2-, 4-, 8-, 16-, 32-cell and morula or blastocyst stage in co-culture with porcine cumulus cells and mouse fetal fibroblast cells were 61.1~67.0%, 59.0~58.0%, 42.5~43.1%, 28.4~30.2% and 20.4~21.0%, respectively. These development rates upto morula or blastocyst stages were significantly higher than those of the embryos cultured in the basic culture medium(P<0.01). These findings suggest that co-culture of in vitro fertilized eggs with porcine cumulus cells or mouse fetal fibroblast cells enhance the development of fertilized eggs to morula or blastocyst stage in vitro.

• Proceedings of the KSAR Conference
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• 2003.06a
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• pp.62-62
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• 2003

This study was carried out to investigate the effects of activation agents on parthenogenetic activation of pig oocytes matured in vitro. The medium used for oocyte maturation was tissue culture medium (TCM) 199 supplemented with 26.19 mM sodium bicarbonate, 0.9 mM sodium pyruvate, 10 $\mu\textrm{g}$/ml insulin, 2 $\mu\textrm{g}$/ml vitamin $B_{l2}$, 25 mM Hepes, 10 $\mu\textrm{g}$/ml bovine apotransferrin, 150 $\mu$M cysteamine, 10 IU/ml PMSG, 10 IU/ml hCG, 10 ng/ml EGF, 0.4% BSA, 75 $\mu\textrm{g}$/ml sodium penicillin G, 50 $\mu\textrm{g}$/ml streptomycin sulfate and 10% pFF. After about 22 h of culture, oocytes were cultured without cysteamine and hormones for 22 h at 38.5$^{\circ}C$, 5% $CO_2$ in air. Cumulus-free oocytes involving first polar body were activated by exposure to various concentrations of ethanol and exposure time of ethanol in Hepes-buffered NCSU23 medium. Also, oocytes were activated by electric pulse alone or combination with ethanol. For electrical activation, oocytes were rinsed twice in 0.3 M mannitol solution supplemented with 0.1 mM CaC1$_2$, 0.2 mM MgC1$_2$, 0.5 mM Hopes and 0.01% BSA, and transferred to a chamber consisting of two electrodes 1 mm apart which was overlaid with the same activation solution. Oocytes were activated with a single DC pulse of 1.3 ㎸/cm for 30 $\mu$sec. After activation treatments, oocytes were washed three times with Hepes-buffered NCSU23 medium and were washed twice with NCSU23 culture medium containing 0.4% BSA, and then cultured in 500 ${mu}ell$ of the same medium for 20 h at 38.5$^{\circ}C$, 5% $CO_2$ in air. The activation rates of oocytes were higher in 6, 7 and 8% ethanol concentrations compared with 0, 5, 9 and 10% ethanol concentrations. Significantly more oocytes (29.3~33.7%) were activated in the exposure for 8, 10, 12 and 15 min than those in the exposure for 0 and 5 min, but there was no difference due to exposure to 8% ethanol for 8 to 15 min. Electric pulse treatment followed by exposure to ethanol significantly improved the rate of oocyte activation (61.9%) compared with that of other 3 treatments. In conclusion, the optimal activation treatment of ethanol exposure alone for the in-vitro matured pig oocytes was 8% ethanol for 8 to 15 min. Electric pulse treatment followed by ethanol exposure significantly improved the rate of activation.n.

• Asian-Australasian Journal of Animal Sciences
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• v.21 no.4
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• pp.494-498
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• 2008

MTT (3-(4, 5-dimethyl thiazol-2-yl)-2, 5-diphenyl tetrazolium bromide) reduction assay is a method that validates the viability of an active cell. Dehydrogenase in mitochondria converts yellow colored insoluble tetrazolium salt to purple colored water-soluble formazan. Sperm also have mitochondria in the midpiece, therefore sperm viability could be evaluated by MTT reduction assay. Several studies have already demonstrated the capability of application of the MTT reduction assay to sperm of several species in Hepes-BSA buffer. Because most liquid semen was diluted in extender like BTS (Beltsville Thawing Solution), Modena or Androhep when it is used or transferred, semen needed another dilution in Hepes-BSA buffer to assess sperm viability. In this study, we evaluated boar sperm viability especially in BTS extended semen and compared the efficiency of this test with eosin-nigrosin staining. We used the fresh BTS extended semen from a local A.I center. Semen sample was diluted to $3.0{\times}10^7$ sperms/ml in BTS. The rates of formazan production were measured in 96-well microtiter plates immediately and 1h after incubation at $17^{\circ}C$ using a spectrophotometer at wave length 560 nm. Simultaneously, split samples of the same semen were tested, using eosin-nigrosin staining to compare the efficiency of the MTT assay of sperm viability in BTS. The correlation between the results of these tests was calculated using Student-t test and ANOVA. The results revealed a strong correlation between the results of MTT reduction rate and the results that were simultaneously determined by eosin-nigrosin staining at 1 h. In conclusion, the MTT reduction test was an effective and simple method to validate sperm viability and it could be used as a simple tool to evaluate sperm viability in the local A.I center and laboratory.

• Applied Chemistry for Engineering
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• v.8 no.4
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• pp.660-666
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• 1997

The production of doxorubicin by a mutant of Streptomyces peucetius subsp. caesius was studied. The optimal culture conditions, such as inoculum size and medium composition were established to improve the productivity of doxorubicin. The optimal medium composition was found to be 4% maltose, 0.5% HEPES, 0.02% $K_2HPO_4$, 0.01% $MgSO_4$. As an antiform agent, 0.001% KG(10% Adekanol+10% Silicone) was suitable one among various agents. Culture was carried out in 2.5 L jar-fermenter with different aeration rates of 1.5, 1.0, and 1.5 v/v min. The maximum production of doxorubicin(29 mg/l) was obtained at 1.5 v/v min of aeration rate, and even at 1.0 v/v min, the production of doxorubicin was increased up to 15% compared with that of shake-flask culture.

• Journal of Pharmaceutical Investigation
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• v.34 no.6
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• pp.491-497
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• 2004

We investigated some important factors which affect the transdermal flux of ketoprofen, a nonsteroidal anti-inflammatory agent, as a first step to provide some basic knowledge for the development of a iontophoretic transdermal patch system. Factors such as current density, polarity, buffer (HEPES) and electrolyte concentration and pH were studied using hairless mouse skin. The effect of poly(L-lysin), which is known to affect the electro-osmotic flow through skin, on flux was also studied. Passive flux was about $20\;{\mu}g/cm^2hr$ at pH 4.0, but was negligible at pH 7.4 where all ketoprofen molecules dissolved are ionized (ketoprofen pKa=5.94). At pH 4.0, application of anodal current increased the flux further above the passive level, however anodal flux at pH 7.4 was much smaller than passive flux at pH 4.0. The application of cathodal current at pH 4.0 increased the average flux to $30-40\;{\mu}g/cm^2hr$, depending on the current density applied. At pH 7.4, cathodal flux was only about $5\;{\mu}g/cm^2hr$. Decrease in buffer and electrolyte concentration increased this cathodal flux about 10 fold. However decrease in HEPES buffer concentration 100 fold did not affect the flux. Anodal flux of acetaminophen was much larger than cathodal flux, indicating that electroosmotic flow can be playing an important role in the flux. Poly(L-lysin) increased the cathodal flux at pH 7.4. These results provide some important insights into the mechanism of transdermal flux of ketoprofen and the role of electroosmotic flow.

• Applied Chemistry for Engineering
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• v.34 no.3
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• pp.279-284
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• 2023

Isothermal titration calorimetry (ITC) is a useful technique to obtain thermodynamic binding properties such as enthalpy, Gibbs free energy, entropy, and stoichiometry of the chelation reaction. A single independent binding site model was used to evaluate the thermodynamic binding properties in BaCl₂ and ethylenediaminetetraacetic acid (EDTA) in Trince and HEPES buffers. ITC enables us to elucidate the binding mechanism and find an optimal chelation condition for BaCl₂ and EDTA in the pH range of 7~11. Chelation of BaCl₂ and EDTA is a spontaneous endothermic reaction. As pH increased, entropic contributions dominated. The optimal pH range is narrow around pH 9.0, where 1:1 binding between BaCl₂ and EDTA occurs.

• Journal of Chest Surgery
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• v.24 no.9
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• pp.837-842
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• 1991

Calcification is a major problem in glutaraldehyde-preserved bioprosthetic valves. We have used bovine pericardium processed in a solution containing 0.625% glutaraldehyde, 0.05M HEPES buffer and 0.26% magnesium chloride in saline. And, we also treated the glutaraldehyde-preserved bovine pericardium with a surfactant, Triton X - 100 to reduce calcification. To evaluate the degree of calcification. 4 kinds of pericardial xenografts, group I [Xenomedica, equine pericardial xenografts], group II [0.625% glutaraldehyde-preserved bovine pericardiums], group III [0.5% Triton X - 100 treated bovine pericardiums], and group IV [1.2% Triton X - 100 treated bovine pericardiums] were implanted in subcutaneous layer of growing rabbits, and they were explanted about 3 months later. The mean calcium contents[%/mg of dry tissue] of 0.5% and 1.2% Triton X - 100 treated bovine pericardiums [80.0$\pm$27.1%: 78.6$\pm$47.0% respectively] were lower than those of glutaraldehyde-preserved bovine pericardiums[126.2$\pm$29.8] [p=0.05]. Thus, under the conditions of subcutaneous implantation in rabbits, Triton X - 100 was efficient in calcification mitigation.

• Bulletin of the Korean Chemical Society
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• v.32 no.11
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• pp.3959-3962
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• 2011

A simple propargylamide-fuctionalized chemodosimeter was prepared for the ratiometric fluorescence detection of mercuric ions in HEPES buffer. The chemodosimeter exhibited $Hg^{2+}$-induced propargyl amide-tooxazole transformation, with a significant accompanying ratiometric change in fluorescence. It afforded high selectivity for mercuric ion detection without any competitive inhibition by common alkali, alkaline earth, or other transition metal ions. The probe showed a $17{\times}10^{-6}M$ detection limit for $Hg^{2+}$ ions and potential applicability for detecting aqueous $Hg^{2+}$ ions.

• Korean Journal of Animal Reproduction
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• v.16 no.1
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• pp.39-46
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• 1992

In viro fertilizatin is very important in both human clinical practice and animal breeding. However, the success rate of in vitro fertilization is not high. The purpose of this study ws to determine wheter in the vitro fertilization and culture of porcine oocyte using a hydrogel chamber were possible or not. Hydrogel chambers were made of polymerized 2-hydroxyethyl methacrylate. Matured follicular oocytes in Waymouth's medium and T L Hepes medium, tubal oocytes, and preincubated sperm in M199 medium were treansferred into the lumen of the hydrogel chambers. The chambers containing porcine oocytes and spermatozoa implanted into the mouse peritioneal cavity, and ova were examined after the recovery of the chambers at 84 hours after preservation start. The result was shown that fertilization and culture of porcine oocytes were successfully achieved inside of the hydrogel chamber.

• Microbiology and Biotechnology Letters
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• v.21 no.4
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• pp.381-391
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• 1993

As the first stage of development of an artificial skin, fibroblasts were cultured in the collagen matrices to make a living dermal equivalent. Mouse embryonic fibroblasts were incorporated into a collagen matrices on plastic dishes containing concentrated DMEM culture media supplemented with sodium bicarbonate, hepes, antibiotics and fetal bovine serum. As the growth stimulation components, glycosaminoglycans were added: hyaluronic acid, chondroitin sulfate, heparin, chitosan were incorporated into the media at a concentration of either 1% or 5% w/w/ to collagen in order to investigate the effect on development of dermal equivalent. After the few days of incubation, gel matrics were contracted and firm dermal equivalent were formed. And the keratinocytes were cultured on top of dermal equivalent and make a three dimensional artificial skin tissue.