• 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.

• Reproductive and Developmental Biology
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• v.30 no.1
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• pp.53-58
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• 2006

The study was conducted to evaluate the effects of culture period and fusion method on the development of somatic cell nuclear transfer (SCNT) embryos reconstituted with Korean bovine fetal fibroblast cells (KbFF) and Korean bovine adult ear skin fibroblast cells (KbESF). KbFF were isolated from a day 51 Korean cattle (Hanwoo) fetus, and KbESF were isolated from a 28 month old Hanwoo calf. The cells were cultured up to 15 weeks (passage 15) in vitro for SCNT. Chamber and electrode needles were used for comparing fusion of reconstituted eggs. The doubling times of KbFF and KbESF were 17.3 hr and 24.3 hr, respectively. The fusion and cleavage rates were significantly higher in needle group (76.1 and 81.2% respectively, P<0.05) than those in chamber group. However, the blastocyst development rate was not different between both groups. Fusion and cleavage rates of NT eggs reconstituted with KbESF did not affected by passage number, however, blastocyst rates were lower in passage $1{\sim}4$ group (21.3%) than passage $5{\sim}8$ (39.4%) and $13{\sim}15$ groups (40.4%, P<0.05). Whereas, fusion rate was lower in passage $1{\sim}4$ group (61.5%) than those of passage $5{\sim}8$(75.0%) and $13{\sim}15$ (76.8%) groups, but cleavage and blastocyst rates were similar regardless of passage number in the KbFF. The results suggest that fusion method can affect the development of SCNT embryos, whereas the long term culture up to 15 passages may not affect the development of SCNT embryos.

• Asian-Australasian Journal of Animal Sciences
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• v.29 no.6
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• pp.845-854
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• 2016

Characteristics and properties of gelatin from goat skin pretreated with NaOH solutions (0.50 and 0.75 M) for various times (1 to 4 days) were investigated. All gelatins contained ${\alpha}$-chains as the predominant component, followed by ${\beta}$-chain. Gelling and melting temperatures of those gelatins were $23.02^{\circ}C$ to $24.16^{\circ}C$ and $33.07^{\circ}C$ to $34.51^{\circ}C$, respectively. Gel strength of gelatins increased as NaOH concentration and pretreatment time increased (p<0.05). Pretreatment for a longer time yielded gelatin with a decrease in $L^*$-value but an increase in $b^*$-value. Pretreatment of goat skin using 0.75 M NaOH for 2 days rendered the highest yield (15.95%, wet weight basis) as well as high gel strength (222.42 g), which was higher than bovine gelatin (199.15 g). Gelatin obtained had the imino acid content of 226 residues/1,000 residues and the gelatin gel had a fine and ordered structure. Therefore, goat skin gelatin could be used as a potential replacer of commercial gelatin.

• Environmental Mutagens and Carcinogens
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• v.18 no.2
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• pp.98-103
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• 1998

Epigallocatechin gallate (EGCG) was reported to exert weak cytotoxicity against normal healthy cells such as C3H10T1/2 cells, but profound inhibitory effects on the initiation or promotion stage of chemical carcinogenesis in mammary gland, blood and mouse skin. This study was carried out to develop antitumor agents with weak side effects and strong antitumor activity. Human skin melanoma cells (HBT 69) and human oral epitheloid carcinoma cells (OCL 17) were cultured in RPMI-1640 media containing 10% fetal bovine serum, antibiotic, and fungizone. After incubation for 24 hrs, the cells were treated with various amounts of (EGCG) for 48 hrs. The growth inhibitory effects of EGCG in human oral epitheloid carcinoma cells were evaluated by the 3- (4,5-djmethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), neutral red (NR), and sulforhodamine B protein (SRB) assays of colorimetric methods. The light microscopic study was also carried out to observe morphological changes of the treated cells. These results obtained were as follows; 1. Significantly inhibitory effects of EGCG against cultured human oral epithelioid carcinoma cells. 2. Significantly inhibitory effects against cultured human skin melanoma cells treated with 50 $\mu$M EGCG, but decreased inhibitory effects in 100 $\mu$M EGCG. 3. Degenerative changes against cultured human oral epitheloid carcinoma cells. 4. Degenerative changes against human skin melanoma cells treated with 50 UM EGCG, but recovered degenerative changes in 100 $\mu$M EGCG.

• Journal of Veterinary Clinics
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• v.4 no.2
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• pp.463-472
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• 1987

Although there are many reports on the bovine electroacupuncture anesthesia, its application to clinical cases needs much to be verified. In order to apply the electroacupuncture anesthesia to bovine species, a new anesthesia technique, which is safe and effective, must be developed. In the present study, 11 meridian points were selected and 11 kinds of meridian points were prescribed to develop an effective and safe electroacupucture methods in the bovine species. The results obtained are summarized as fellows : 1. When the maximun electric current (30Hz at 5.3-5.6V ) was applied, anesthesia was attained with the signs of tetany and tremor : When the electric current was continued for 15-20 minutes, the signs of tetany and tremor diminished markedly. 2. If the electric current was adjusted to 30Hz at 3.5-4.0V and continued, the tetany and tremor disappeared. The skin was slackened, thus suitable for surgical operation. The continuation of anesthesia could be regulated at operator's own will. 3. The same anesthesia effect could be attained by using any one meridian point when the points were symmetrical. 4. Transient changes of clinical signs and blood pictures were noted during the stages of acupuncture anesthesia. However, the red blood cell counts, white blood cell counts and hematocrit values were always within the normal range.

• Journal of Veterinary Clinics
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• v.2 no.1
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• pp.79-104
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• 1985

Clinical and cytomorphological studies were carried out in 32 leukotic cattle from Tokachi and Kushiro districts in Hokkaido during the 12 year period from 1969 to 1980. The leukotic cattle were examined :and divided into four types(15 cases of the adult, 11 cases of the thymic, 4 casas of the calf and 2 cases of the skin types). The results obtained were as follows : 1. As for the frequency of the main clinical signs in each type, In the adult type, the main clinical signs (of decreasing order) are as follows: swelling of the superficial lymph nodes>depression and loss of weight>tachycardia>anorekia, anemia of the visible mucous membrane and tachypnea. Those of the thymic type were swelling of the thymus>swelling of the medial iliac lymph nodes> swelling of the superficial lymph nodes>tachypnea. Those of the calf type were swelling of the auperficial lymph nodes>depression and emaciation>tachypnea>anorexia, tachycardia, anemia of the visible mucous membrane and recumbency. Those of the skin type were generalized urticaria-like lesions in skin and swelling of superficial lymph nodes>and depression and loss of weight in the decreasing order of frequency. In addition, large tumor mass in the pelvic cavity and swelling of the medial iliac lymph nodes were detected through rectal palpation in 33.3% and 100% in the adult type cases, respectively. 2. As for the hematological findings, The frequency of occurrence of decreased erythrocyte counts in the decreasing order were as follows : adult>calf>thymic>and skin types. The increase in the total leukocyte count in the order of decreasing frequency were as follows: calf>thymic>adult>and skin types. The increase in the absolute lymphocyte counts was found to be at a low rate, 62.5% of all the cases examined. By contrast, the increase of 5% or more of abnormal lymphocyte rates was observed at a high rate, 96.9% of the total cases. 3. Abnormal lymphocytes were found in all cases examined for lymph nodes biopsied. 4. From the cytomorphological point of view, leukotic cells were divided into 3 types: reticulum cell, lymphoid cell and monocytic cell types. The adult type leukotic cattle were divided with reticulum cell type (66.7%), the lymphoid cell type(22.6%) and monocytic cell type(6.7%). The thymic type was lymphoip cell type(72.7%) and reticulum cell type(27.3%). In the calf type, all were lymphoid cell type while all of the skin type were reticulum cell type only. 5. The leukotic cattle had higher NP frequency in the blood and lymphoid tissue than non-1 eukotic cattle. Especially the adult type had the highest NP frequency. However, it was not recognized that NP were characteristic of leukotic cattle alone. 6. The above findings lead to the conclusion that the most effective diagnostic methods for bovine leukosis are the confirmation of swelling of the superficial and internal lymph nodes and thymus in addition to appearance of abnormal neoplastic cell in the peripheral blood and lymph nodes biopsied.

• Archives of Plastic Surgery
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• v.40 no.1
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• pp.11-18
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• 2013

Background To minimize the inflammatory reaction and improve healing, a new modified dermal substitute composed of an atelocollagen, chondroitin-6-sulfate, and amniotic membrane (AM) was applied to full-thickness skin defects in a pig. Atelocollagen was extracted from bovine skin, and two modified dermal substitutes were generated according to the cross-linking type. Methods The AM-collagen dermal substitutes were characterized and compared with currently used dermal substitutes in a pig skin defect model. There were five experimental groups: dehydrothermal (DHT) cross-linking atelocollagen with the AM on the top (AM-DHT), DHT and chemical cross-linking atelocollagen with the AM on the top (AM-DHT/chemical), Terudermis, Integra, and AlloDerm. After $3{\times}3cm$ full-thickness skin defects on the back of a pig were created, each dermal substitutes dermal substitutes was randomly grafted on the defects. Two weeks after grafting, autologous partial-thickness skin was over-grafted on the neodermis. The take rate of the dermal substitutes, skin, and histological sections were all assessed at 1, 2, and 4 weeks postoperatively. Results More rapid healing and a higher take rate were evident in the AM-DHT and Terudermis groups. Histological examination revealed fewer inflammatory cells and more fibroblast hyperplasia in these two groups. Four weeks after surgery, the amount of newly formed collagen was significantly more appropriate in the AM-DHT group. Conclusions These observations provide supporting evidence that a newly developed amniotic-collagen dermal substitute may inhibit inflammatory reactions and promote wound healing.

• Proceedings of the Korean Society of Developmental Biology Conference
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• 2003.10a
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• pp.86-86
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• 2003

Research has been in progress for more than a decade to production of useful proteins by genetic modification in cattle. However, the levels of protein production in transgenic cattle have been reported very low. To enhance protein production in transgenic animal, we tried homologous recombination to donor cells for production of transgenic clone cattle through nuclear transfer procedure. Thus, we constructed the two targeting vectors of human thrombopoietin (TPO) at bovine $\beta$-casein locus using homologous recombination with 13.6 kb and 9.6 kb homology. In two targeting vectors, positive selection was through the neomycin resistance gene and negative selection was by the diphtheria toxin (DT). Gene targeting was attempted in bovine embryonic fibroblasts (bEF) and bovine ear skin fibroblasts (bESF). To determine the most appropriate concentration of neomycin for bEF and bESF, G4l8 resistance was confirmed by culturing the cells in various concentrations of the drug and both of the cells were optimally selected at $900 \mu g/ml$ of neomycin. The transfected bEF and bESF by the targeting vectors were colonized efficiently at the ratio of DNA to transfection reagent such as $4 \mu g$:2 ${mu}ell$ and $1 \mu g$:$2 \mu l$. Comparing number of healthy clones from passage 4 to passage 8, bESF (17%) persist in culture for much longer than bEF (6%). The two gene-targeted bESF clones of 30 random-integrated clones with 9.6 kb homology length were confirmed, however, nothing was out of 72 random integration clones with 13.6 kb homology length, The DT also worked more efficiently in clones transfected with the vector of 9.6 kb homology length. Our data suggests that the choice of donor cell for long culture period should be considered to obtain targeted cell clone, and the gene-targeting frequency and the DT working efficiency are dependent on the length of target homology.

• Proceedings of the KSAR Conference
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• 2001.03a
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• pp.69-69
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• 2001

The cell cycle phase in which donor nuclei exist prior to nuclear transfer is an important factor governing developmental rates of reconstituted embryos. It was suggested that quiescent G0 and cycling G1 cells could support normal development of reconstituted embryos. In a quest of optimized donor nuclei treatment prior to nuclear transfer, this study was undertaken to examine the cell cycle characteristics of bovine fetal and adult somatic cells when cultured under a variety of culture treatments and the cell cycle change with the lapse of time after trypsinization. This was archived by measuring the DNA content of cells using flow cytometry, Cultured fetal fibroblast cells, adult skin and muscle cells, and cumulus cells were divided by 3 culture treatments; 1) grown to 60-70% confluency (cycling), 2) serum starved culture, 3) culture to confluency. Trypsinized cells were fixed by 70% ethanol and stained with propidium iodide. For one experiment, trypsinized cells were resuspended in DMEM＋10% FBS and incubated for 1.5, 3 and 6 h with occasional shaking before ethanol fixation. Cell cycle phases were determined by flow cytometry enabling calculation of percentages of G0＋G1, S and G2＋M. The majority of cells were in G0＋Gl stage regardless of origin of cells. Cultures that were serum starved or cultured to confluency contained significantly (P<0.05) higher percentages of cells in G0＋G1 (89.5-95.4%). For every cell lines and culture treatments, percentages of cells in existing in G0＋G1 increased with decreasing of the cell size from large to small. In the serum starved and confluency groups, about 98% of small cells were in G0＋G1 Serum starved culture contained higher percentages of small-sized cells (38.5-66.9%) than cycling and confluent cultures regardless of cell lines (P<0.05). After trypsinization of fetal fibroblast and adult skin cells that were serum starved and cultured to confluency, the percentages of cells in G0＋G1 significantly increased by incubation for 1.5(95.7-99.5%) and 3.0 h (95.9-98.6%). The results suggest that the efficient synchronization of bovine somatic cells in G0＋G1 for nuclear transfer can be established by incubation for a limited time period after trypsinization of serum starved or confluent cells.

• Reproductive and Developmental Biology
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• v.35 no.4
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• pp.485-489
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• 2011

The present study was conducted to examine the reactive oxygen species (ROS) generation levels and subsequent DNA damage in the bovine cultured somatic cells. Bovine ear skin cells were classified by serum starvation, confluence and cycling cells. Cells were stained in 10 ${\mu}M$ dichlorohydrofluorescein diacetate ($H_2DCFDA$) or 10 ${\mu}M$ hydroxyphenyl fluorescein (HPF) dye to measure the $H_2O_2$ or $^{\cdot}OH$ radical levels. The samples were examined with a fluorescent microscope, and fluorescence intensity was analyzed in each cell. $H_2O_2$ and $^{\cdot}OH$ radical levels of cultured somatic cells were high in confluence group ($7.1{\pm}0.7$ and $8.4{\pm}0.4$ pixels/cell, respectively) and significantly low in serum starvation group ($4.9{\pm}0.4$ and $7.0{\pm}0.4$ pixels/cell, respectively, p<0.05). Comet tail lengths of serum starvation ($148.3{\pm}5.7$ ${\mu}M$) and confluence ($151.1{\pm}5.0$ ${\mu}M$) groups were found to be significantly (p<0.05) increased in comparison to that of cycling group ($137.1{\pm}7.5$ ${\mu}M$). These results suggest that the culture type of donor cells can affect the ROS generation, which leads the DNA fragmentation of the cells.