• Korean Journal of Animal Reproduction
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• v.11 no.3
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• pp.155-160
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• 1987

This study was carried out to determine the effects of cryoprotectants, freezing and thawing rates on the survival of 8-cell mouse embryos. The female ICR mice were induced to superovulated by intraperitoneal injections of 5 i.u. PMSG and 5 i.u. HCG given 48h apart and then were paired with males of the same strain. They were killed and embryos were flushed from the oviducts and uteri on 3 days after injection of HCG. Embryos were flushed with modified Dulbecco's phosphate buffered saline and equilibrated with 1.5M-dimethyl sulfoxide (DMSO) or 1.5M-glycerol by 3-step procedure. The freezing rates of the embryos were 1$^{\circ}C$/min from room temperature to -5$^{\circ}C$ and the embryos were seeded at -5$^{\circ}C$. After being held for 3 min at the seeding temperature, the rates were 0.3$^{\circ}C$/min from -5$^{\circ}C$ to -35$^{\circ}C$. From -35$^{\circ}C$ to -7$0^{\circ}C$, the rates were divided into 0.1$^{\circ}C$/min, 1$^{\circ}C$/min and 1$0^{\circ}C$/min, respectively. After being held for 5 min at -7$0^{\circ}C$, the embryos were plunged directly into liquid nitrogen. The embryos were thawed at 4$^{\circ}C$/min and 12$^{\circ}C$/min from -196$^{\circ}C$ to 37$^{\circ}C$, and for 2 min in 37$^{\circ}C$ water bath, respectively. The average number of ovulation points and embryos recovered were 42.7 and 34 appearing 79.5% recovery rate. Eight cell embryos in the embryos recovered were 26.3. The survival rates of embryos according to the freezing rates in the presence of 1.5M-DMSO were 73.5~80.6% at 0.1$^{\circ}C$/min, 75.0~79.5% at 1$^{\circ}C$/min and 52.8~54.7% at 1$0^{\circ}C$/min, but in the presence of 1.5M-glycerol were 62.9~67.6% at 0.1$^{\circ}C$/min, 61.4~68.3% at 1$^{\circ}C$/min and 25.5~30.2% at 1$0^{\circ}C$/min. The survival rates of embryos were not affected by the thawing rates.

• Archives of Plastic Surgery
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• v.35 no.6
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• pp.645-652
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• 2008

Purpose: In skin flap surgery, surgeons often encounter distal ischemia of the flap. If a powerful free radical scavenger is used, it may reduce the formation of free radical and improves the survival of flap. Thus, the present study purposed to examine whether the survival of flap can be enhanced by administering melatonin, which is known to be a powerful free radical scavenger a antioxidant molecule. Methods: We divided 40 Sprague-Dawley rats into 4 groups, 10 in each group. For the control group(n=10), we intraperitoneally injected only carrier solution once 30 minutes before the operation, and once a day for 7 days from the day of operation. Among the experimental groups, a group(n=10) was administered with dimethyl sulfoxide(DMSO), in another group(n=10), melatonin was intraperitoneally injected, and in the other(n=10) melatonin was intraperitoneally injected and applied topically(2 cc of 1% melatonin) to the operation site. Caudally based skin flaps measuring $3{\times}10cm^2$ were elevated on the mid-dorsum of the rats. and then repositioned. On the seventh postoperative day, the survival area of the flap was measured and tissues were examined under the light microscope. Results: The control group, the DMSO group, the melatonin administration group and the melatonin administration and application group showed the mean survival rates of $55.26{\pm}9.2%$, $70.29{\pm}7.47%$, $81.45{\pm}4.14%$ and $86.1{\pm}1.52%$, respectively, for $30cm^2$ of flap. Compared to the control group, the experimental groups showed a significantly high increase in survival area at significance level of 95%. Conclusion: In this study, the survival rate of flap was enhanced through the administration of melatonin after flap surgery. This suggests that melatonin not only functions as a powerful free radical scavenger and oxygen radical scavenger but also stabilizes and protects cells, and by doing so, enhances the survival of moderately injured ischemic sites in the distal end of flap.

• Development and Reproduction
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• v.13 no.3
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• pp.173-181
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• 2009

One of the most extensively studied populations of multipotent adult stem cells are mesenchymal stem cells (MSCs). MSCs derived from the human umbilical cord vein (HUC-MSCs) are morphologically and immunophenotypically similar to MSCs isolated from bone marrow. HUC-MSCs are multipotent stem cells, differ from hematopoietic stem cells and can be differentiated into neural cells. Since neural tissue has limited intrinsic capacity of repair after injury, the identification of alternate sources of neural stem cells has broad clinical potential. We isolated mesenchymal-like stem cells from the human umbilical cord vein, and studied transdifferentiation-promoting conditions in neural cells. Dopaminergic neuronal differentiation of HUC-MSCs was also studied. Neural differentiation was induced by adding bFGF, EGF, dimethyl sulfoxide (DMSO) and butylated hydroxyanisole (BHA) in N2 medium and N2 supplement. The immunoreactive cells for $\beta$-tubulin III, a neuron-specific marker, GFAP, an astrocyte marker, or Gal-C, an oligodendrocyte marker, were found. HUC-MSCs treated with bFGF, SHH and FGF8 were differentiated into dopaminergic neurons that were immunopositive for tyrosine hydroxylase (TH) antibody. HUC-MSCs treated with DMSO and BHA rapidly showed the morphology of multipolar neurons. Both immunocytochemistry and RT-PCR analysis indicated that the expression of a number of neural markers including NeuroD1, $\beta$-tubulin III, GFAP and nestin was markedly elevated during this acute differentiation. While the stem cell markers such as SCF, C-kit, and Stat-3 were not expressed after neural differentiation, we confirmed the differentiation of dopaminergic neurons by TH/$\beta$-tubulin III positive cells. In conclusion, HUC-MSCs can be differentiated into dopaminergic neurons and these findings suggest that HUC-MSCs are alternative cell source of therapeutic treatment for neurodegenerative diseases.

• Reproductive and Developmental Biology
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• v.30 no.1
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• pp.47-52
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• 2006

This study was conducted to examine whether the parthenogenetic mouse embryonic stem (P-mES) cells can differentiate into functional cardiomyocytes in vitro similar to (mES) cells. p-mES04 and IVF-derived mES03 cells were cultured by suspension culture for 4 days. The formed embryoid bodies (EBs) were treated with 0.75% dimethyl-sulfoxide (DMSO) for further 4 days (4-/4+), and then plated onto gelatin coated culture dish. The appearance of contracting cardiomyocytes from the P-mES04 and mES03 cells was examined for 30 days. The highest cumulative frequency was detected at days 13 (69.83%) and 22 (61.3%), respectively. By immunocytochemistry, beating P-mES04 cells were positively stained with muscle specific anti-sarcomeric a-actinin Ab and cardiac specific anti-cardiac troponin I Ab similar to contracted mES03 cells. When the expression of cardiac muscle-specific genes was analyzed by RT-PCR, beating P-mES04 cells were expressed cardiac specific L-type calcium channel, a1C, cardiac myosin heavy chain a, cardiac muscle heavy polypeptide $7{\beta}$, GATA binding protein 4 and atrial natriuretic factor, but not expressed skeletal muscle specific L-type calcium channel, a1S, which was similar to male adult heart cells and mES03-derived beating cardiomyocytes. The result demonstrates that the P-mES cells can be used as an alternative for the study on the characteristic analysis of in vitro cardiomyocyte differentiation from the ES cells.

• Journal of the East Asian Society of Dietary Life
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• v.24 no.6
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• pp.776-784
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• 2014

Korean dishes, Hansik are characterized by healthful vegetable intake. The purpose of this study was to evaluate the inhibitory effect of commonly consumed vegetables by Koreans on obesity/metabolic disease-related inflammation. Through statistical analysis of the KNHANES database ($1^{st}$ 1998, $5^{th}$ 2010, 2011) and a literature review, we selected vegetables for study. Among the vegetables, main or sub ingredients of Kimchi were excluded. Samples were prepared using only edible portions and freeze-dried. After grinding, samples were extracted with ethanol, evaporated and finally lyophilized. The cytotoxicity of samples was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, at various concentrations that do not affect cell viability. Raw 264.7 macrophages were treated with lipopolysaccharide (LPS) and 11 kinds of samples or positive control (troglitazone) dissolved in dimethyl sulfoxide (DMSO). After 24 hours, nitric oxide (NO), tumor necrosis factor ${\alpha}$ (TNF-${\alpha}$) and monocyte chemoattractant protein-1 (MCP-1) production were determined. Excepts for young pumpkin and bracken, nine samples effectively reduced NO production compared with control treated with LPS and DMSO. NO levels of five samples (bean sprouts, leeks, eggplant, mugwort, and pumpkin) were similar to that of the positive control. These five samples showed significantly decreased TNF-${\alpha}$ or MCP-1 compared to the control group. Our results suggest that consumption of commonly consumed vegetables contributes to partial prevention of obesity and related metabolic syndrome through reduction of NO, TNF-${\alpha}$, and MCP-1 production.

• Proceedings of the Korean Society of Developmental Biology Conference
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• 2003.10a
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• pp.72-72
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• 2003

The present study examined the possibility of cryopreservation of the D-shaped and umbo larvae of arkshell (Scapharca broughtonii), in terms of the survival rates after freezing and thawing. D-shaped and umbo larvae of arkshells were obtained from a shellfish farming on Yosu city. The average shell lengths were $93.3 \pm 10.1 \mu$m and $201.7 \pm 13.5 \mu$, respectively. Five cryoprotectants (CPAs), dimethyl sulfoxide (DMSO), glycerol, ethylene glycol (EG), propylene glycol (PG), and methanol, were tested at the concentrations of 1.5, 2.0 and 2.5 M. After larvae suspended in CPAs, cryoprotectants were loaded in 0.5 ml straws at a larval density of 50-100 larvae per straw, and epuilibrated for 10 and 20 minute at room temperature ($23^{\circ}C$), repectively. Straws were cooled at a rate of $1^{\circ}C$/min from $0^{\circ}C$ to $-12^{\circ}C$, held for 5 min at $-12^{\circ}C$, and then cooled at $2^{\circ}C$/min to $-35^{\circ}C$ and equilibrated for 5 min followed by plunging in liquid nitrogen. After storage in liquid nitrogen for 1 day, straws were thawed in a $30^{\circ}C$ water. As soon as straws were observed to melt, larvae were diluted with an equal volume of ASW and then washed twice with a large volume of ASW at an interval of 2 min to unload the CPAs. The results showed that after equilibration for 10 and 20 minute at room temperature, no larvae survived using methanol as CPAs, and it was observed that larval shells all open slightly, and larval flesh broke down and slopped over the shells. The highest survival rates (D-shaped larvae: 77.6%, umbo larvae: 59.3%) were obtained with 2M DMSO, and 1.5M glycerol yielded survival rates of 53.8% for D-shaped larvae and 37.5% for umbo larvae. The surviving D-shaped larvae showed active rotary motion and perfect membrane integrity and cytoplasmic normality, and the vigorous movement of veliger cilia was observed inside the closed shells. The breakdown of tissue occurred in the abnormal larvae, and the isolated cell often run out of shells.

• Korean Journal of Environmental Biology
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• v.21 no.1
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• pp.26-30
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• 2003

Cytochrome P450 (CYP) induction was determined in microsomes of three aquacultured fish species (Sebastes schlegeli, Paralichthys olivaceus and Pagrus major) and two wild fish species (Mugil cephalus and Stephanolepis cirrhifey) in vitro exposed to $\beta$-naphthoflavone (BNF). The microsomes of five fish were exposed to BNF (5 mM or 10 mM) in dimethylsulfoxide at $30^{\circ}C$ for 9 hr. The CYP contents in most fish increased according to exposure duration for 3 or 5 hour, and then decreased, while steady increase of CYP was observed in P. major for 9 hour. The induction of CYP contents in aquacultured fish species (207~422%) were higher than those in wild fish species (206~207%).

• Journal of Applied Biological Chemistry
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• v.56 no.3
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• pp.157-163
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• 2013

Cultured products (callus and exopolysaccharide) were obtained from suspension culture of Aloe vera callus, and the extracts of callus were further prepared with cold water or 60% ethanol solution. The ethanol extract of callus (AC) and exopolysaccharide (ACP) of 10 mg/mL exhibited the relatively higher suppression activity of 43.2-52.1% against hyaluronidase activity. Thus, their anti-inflammatory effects were further investigated using animal cell (Raw 264.7) in vitro. Though AC shows a slight suppression effect of cell survival rate (97%) using MTT assay in the presence of $400{\mu}g/mL$ AC- dimethyl sulfoxide (DMSO), cell growth promotion was observed in the other samples of lower levels. It indicates that the ethanol extract of Aloe callus rarely affect cell survival rate in the ranges ($200-400{\mu}g/mL$) used in the study. Using Griess reagent, the suppression of NO production by the aloe callus extract was analyzed by measuring the amount of the nitrite produced in Raw 264.7 culture activated by lipopolysaccharide (LPS). As a result, supplementation of AC-distilled water (DW) and AC-DMSO produced higher levels of NO than the positive control LPS. However, the NO suppression effect by ACP-DW was so intense that lower amount ($80-100{\mu}g/mL$) suppressed NO production to the level of the control. The effect was attributed to the expression of the iNOS. Then, Raw 264.7 cells were stimulated with the LPS and expression of COX-2 protein level was analyzed depending on the Aloe suspension culture product treatment. The results showed that the ACP-DW supplemented medium did not express COX-2 by itself, and LPS stimulated COX-2 expression was slightly decreased. On the other hand, realtime-PCR analysis of the expression of inflammatory cytokine showed that IL-$1{\beta}$ and TNF-${\alpha}$ expression was highly suppressed in the ACP- distilled water supplemented medium.

• Korean Journal of Poultry Science
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• v.41 no.4
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• pp.261-270
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• 2014

This study was conducted to establish the method for preserving chicken primordial germ cells (PGCs) that enables long-term storage in liquid nitrogen ($LN_2$) for developmental engineering or preservation of species. The purpose of this study is to clarify the effects of simple freeze-thaw treatment on viability of PGCs in chickens and to the optimal protocol for PGCs freezing. PGCs obtained from the germinal gonade of an early embryos of 5.5~6 day (stage 28) of Isa Brown, Korean Ogye (KO), White Leghorn and Commercial breeds, using the MACS method were suspended in a freezing medium containing a freezing and protecting agents (e.g. dimethyl sulfoxide (DMSO), ethylene glycol (EG) and propylene glycol (PG)). The gonadal cells, including PGCs, were then frozen in 1 of the following cryoprotectant treatments : 2.5%, 5%, 10%, 15%, and 0% cryoprotectant (DMSO, EG, PG) as a control. Effects of exposure to simple freezing, with different concentrations of the cryoprotectant solution, were examined. After simple freezing, the viability of PGCs after freeze-thawing was significantly higher for Commercial breeds ($88.7{\pm}2.4%$) than KO ($85.1{\pm}0.4%$), Isa Brown ($84.6{\pm}0.2%$) and White Leghorn ($85.9{\pm}0.1%$) (p<0.05) using 10% EG cryoprotectant. Therefore, these systems may contribute in the improvement of cryopreservation for a scarce species in birds preservation. This study established a method for preserving chicken PGCs that enables systematic storage and labeling of cryopreserved PGCs in liquid ($LN_2$) at a germplasm repository and ease of entry into a database.

• Korean Journal of Poultry Science
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• v.40 no.3
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• pp.207-216
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• 2013

This study was conducted to establish the method for preserving PGCs that enables long-term storage in liquid nitrogen for developmental engineering or preservation of species. The purpose of this study is to clarify the effects of freeze-thaw treatment on viability of PGCs in chickens. PGCs were collected separately from a germinal gonad of an early embryo of 5.5~6 day (stage 28) of Isa brown, Korean Oge (KO), White Leghorn and Commercial breeds. PGCs separated from a germinal gonad of an early embryo of 5.5~6 day (stage 28) are suspended in a freezing medium containing a freezing and protecting agents (e.g. dimethyl sulfoxide (DMSO), ethylene glycol (EG) and glycerol). The PGCs were then purified using magnetic activated cell sorting (MACS) method. The viability of PGCs after thawing was $87.4{\pm}0.4%$ and $89.4{\pm}0.2%$ with the 10% EG treatments with no significant difference between the Isa brown and Commercial breeds. The viability of PGCs after freeze- thawing was significantly higher for Isa brown ($87.4{\pm}0.4%$) and Commercial breeds ($89.4{\pm}0.2%$) than Korean Oge (KO) ($77.6{\pm}1.1%$) and White Leghorn ($76.2{\pm}0.9%$)(p<0.05) using 10% EG cryoprotectant. This study established a method for pre- serving chicken PGCs that enables systematic storage and labeling of cryopreserved PGCs in liquid ($LN_2$) at agermplasm repository and ease of entry into a data base. In the future, the importance for this new technology is that poultry lines can be conserved while work is being conducted on improving the production of germline chimeras.