• 한국수정란이식학회지
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• 제11권1호
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• pp.7-14
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• 1996

The object of this study was to evaluate the effect of uterine epithelial cells on development of Korean native cattle(KNC) oocytes fertilized in vitro. Qocytes were collected from ovaries of slaughtered Korean Native Cows and matured in TCM199 with granulosa cells supplemented with 10% FBS, 5$\mu$g/ml FSH, 10 JU/ml hCG, and 1$\mu$g/ml estradiol-17$\beta$ for 24 hrs. For co-culture of in vitro development of fertilized ova, oviductal epithelial cells (l$\times$l0˚cells /ml) obtained from slaughtered cow and uterine epithelial cells(1$\times$10˚cells /ml) flushed from the superovulated holstein on Day 7 were incubated in 39$^{\circ}C$, 5% $CO_2$, 95% air. Frozen-thawed KNC sperm was capacitated with BO(Brackett & Oliphant, 1975) medium supplemented with 10mM, 5mM-caffein. Matured oocytes were inseminated for 20 hrs. And then fertilized oocytes were washed with culture medium and transferred to oviductal epithelial cells for in vitro development and three days later a portion of embryos were transferred to uterine epithelial cells. Stastical methods of developmental rates on KNC-IVF oocytes was ANOVA-test. Developmental rates of KNC-IVF oocytes was significant higher(P<0.01) when co-cul-tured with uterine epithelial cells(25.2%) than oviductal epithelial cells. Blatocyst cul-tured for 7 to 9 days were frozen by automatic freezer with 1.4M glycerol-PBS. Survival rates of blastocyst was 40.0%. Fourteen frozen-thawed blastocysts were transferred to five holstein heifers on day 7 after natural estrus. Three recipients were observed twin and one recipient was single by ultra-sound systems on days 45 after embryo transfer.

• 한국연초학회지
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• 제17권2호
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• pp.109-113
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• 1995

Nicotiana repanda has resistance to many important tobacco diseases but no tobacco cultivars are currently available that carry risistance genes derived from this species. Numerous attempts to hybridize N, repanda with commercial tobacco cultivars have been largely unsuccessful because of cross incompatibility or the uncovering of lethal genes. In vitro pollination of placenta attached ovules was useful in by passing prezygotic barriers for interspecific hybrid combination between N.tabacum cv. NC82 and N. repanda. Six days after in vitro pollination of N. tabacum cv. NC82×N. repanda, enlarged ovules on plancenta were removed and transferred into ovule culture medium of kitsch and kitsch (1969). Within 15 days of ovule culture, germination occurred. Most of the hybrid seedlings obtained had poor root system and finally died, while few of them had good root system and grew well.

• 한국임상수의학회지
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• 제14권2호
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• pp.230-234
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• 1997

In vitro effect of cortisol on the proliferation of canine peripheral blood mononuclear cells (MNC) was examined. The MNC was isolated from peripheral blood by a gradient centrifugation with Picoll-Hypaque. The cell proliferation assayed using a noneradioactive 5-Bromo-2'-deoxy-uridine (BrdU) kit. The MNC proliferated well in response to either phrtobeRagg$]$utinin-p (PHA-P) or culture supernatant from MNC stimulated with PHA-p. However, these proliferative responses of MNC were not affected by addition of coitisol of 1 to 1,OOfl ng/ml. The addition of cortisol in MNC culture with either PHA-P or corture supernatBnt from MNC stimulated with PHA-P far 4 days wag not also influenced on the viabilities of cultured MNC. In conclusions it was able to assay the cell proliferation with BrdU instead of radioactive isotope e.g. tritiated thymidine (3H-TdR). These results suggested that cortisol does not at least influence on MNC proliferation in vitro.

• Journal of Periodontal and Implant Science
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• 제32권1호
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• pp.129-142
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• 2002

Epithelial-mesenchymal interaction plays a important role in cell growth and differentiation. This interaction is already well known to have an importance during the organ development as well as cell growth and differentiation. However, in vitro experimental model is not well developed to reproduce in vivo cellular microenvironment which provide a epithelial-mesenchymal interaction. Because conventional monolayer culture lacks epithelial-mensenchymal interaction, cultivated cells have an morphologic, biochemical, and functional characteristics differ from in vivo tissue. Moreover, it's condition is not able to induce cellular differention due to submerged culture condition. Therefore, the aims of this study were to develop and evaualte the in vitro experimental model that maintains epithelial-mesenchymal interaction by organotypic raft culture, and to characterize biologic properties of three-dimensionally reconstituted oral keratinocytes by histological and immunohistochemical analysis. The results were as follow; 1. Gingival keratinocytes reconstituted by three-dimensional organotypic culture revealed similar morphologic characteristics to biopsied patient specimen showing stratification, hyperkeratinosis, matutation of epithelial architecture. 2. Connective tissue structure was matured, and there is no difference during stratification period of epithelial 3-dimensional culture. 3. The longer of air-exposure culture on three-dimensionally reconstituted cells, the more epithelial maturation, increased epithelial thickness and surface keratinization 4. In reconstitued mucosa, the whole epidermis was positively stained by anti-involucrin antibody, and there is no difference according to air-exposured culture period. 5. The Hsp was expressed in the epithelial layer of three-dimensionally cultured cells, especially basal layer of epidermis. The change of Hsp expression was not significant by culture stratification. 6. Connexin 43, marker of cell-cell communication was revealed mild immunodeposition in reconstitued epithelium, and there is no significant expression change during stratification. These results suggest that three-dimensional oragnotypic co-culture of normal gingival keratinocytes with dermal equivalent consisting type I collagen and gingival fibroblasts results in similar morphologic and immunohistochemical characteristics to in vivo patient specimens. And this culture system seems to provide adequate micro-environment for in vitro tissue reconstitution. Therefore, further study will be focused to study of in vitro gingivitis model, development of novel perioodntal disease therapeutics and epithelial-mensenchymal interaction.

• Reproductive and Developmental Biology
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• 제39권3호
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• pp.49-57
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• 2015

In order to achieve successful in vitro production of embryo, it is necessary to establish intrauterine environment during in vitro culture. Thus, this study was investigated to establish embryo culture system using co-incubated collagen matrix gel (CM) with endometrial epithelial cells (EC). Endometrial epithelial cells were isolated from porcine endometrium at follicular phase, the cells seeded in insert dish for co-incubation with CM-coated culture dish. Then, culture media treated with/without 2.0 IU/ml hCG or 10 ng/ml $IL-1{\beta}$. After incubation for 24 h, the co-incubated insert dishes were removed from CM-coated culture dish before embryo culture. Embryos at 48 h after in vitro fertilization (IVF) were cultured on the dish for 120 h with porcine zygote medium. We determined PTGS-2 expression in the ECs, VEGF protein in co-incubated CM with EC and observed cleavage rate and blastocyst development of embryos at 168 h after IVF. In result, expression of PTGS-2 was higher at co-incubated EC with hCG and $IL-1{\beta}$ groups than EC without hCG and $IL-1{\beta}$. The VEGF protein was detected at co-incubated CM with EC, EC treated with hCG and $IL-1{\beta}$ groups higher than CM group. Also, cleavage rate was no significantly difference among all group, however, blastocyst development was significantly higher in co-incubated CM with EC treated with hCG group than un-treated groups (p<0.05). Therefore, we suggest that novel embryo culture system using co-incubated collagen matrix gel with endometrial epithelial cells treated with $IL-1{\beta}$ is beneficial and useful for enhancing the production of porcine blastocysts in vitro.

• Parasites, Hosts and Diseases
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• 제42권1호
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• pp.27-34
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• 2004

We investigated the optimal culture conditions for Cryptosporidium muris in a human stomach adenocarcinoma (AGS) cell line by determining the effects of medium pH and of selected supplements on the development of C. muris. The optimum pH of the culture medium required for the development of C. muris was determined to be 6.6. The number of parasites significantly increased during cultivation for 72 hr (p < 0.05) at this level. On the other hand, numbers decreased linearly after 24 hr of incubation at pH 7.5. When cultured in different concentrations of serum, C. muris in media containing 5% FBS induced 4-7 times more parasites than in 1% or 10% serum. Of the six medium supplements examined, only 1 mM pyruvate enhanced the number of C. muris in vitro. Transmission electron microscopic observation showed the developmental stages of C. muris in the cytoplasm of the cells, not in an extracytoplasmic location. The growth of C. muris in AGS cells provides a means of investigating its biological characteristics and of testing its response to therapeutic agents. However, a more optimized culture system is needed for the recovery of oocysts on a large scale in vitro.

• 한국수정란이식학회지
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• 제27권2호
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• pp.71-80
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• 2012

Biotechnologies for cloning animals and in vitro embryo production have the potential to produce biomedical models for various researches. Especially, pigs are a suitable model for xenotransplantation, transgenic production and various areas of reproductive research due to its physiological similarities to human. However, utilization of in vitro-produced embryos for transfer remains limited. Despite improvement over past few decades, obstacles associated with the production of good quality embryos in vitro still exist which limit the efficiency of cloning. One of major problems includes improper in vitro maturation (IVM) and culture (IVC). Oxidative stress caused from in vitro culture conditions contributes to inadequate IVM and IVC which leads to poor developmental competence of oocytes, failure of fertilization and embryo development. To reduce the oxidative stress, various antioxidants have been used to IVM and IVC system. However, limited information is available on the effects of resveratrol on livestock reproductions. Resveratrol is a polyphenolic natural product and well known as an antioxidant in foods and beverages (e.g. in grapes and red wine). Resveratrol is known to be cardioprotective, anticarcinogenic, anti-inflammatory, antioxidant and antiapoptotic. This paper will review the effects of resveratrol on in vitro maturation of oocytes and embryo development.

• Journal of Pharmaceutical Investigation
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• 제32권1호
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• pp.21-26
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• 2002

The primary culture of human nasal epithelial cell monolayer was performed on a Transwell. The effect of various factors on the tight junction formation was observed in order to develop an in vitro experimental system for nasal transport studies. Human nasal epithelial cells, collected from human normal inferior turbinates, were plated onto diverse inserts. After 4 days, media of the apical surface was removed for air-liquid interface (ALI) culture. Morphological characteristics was observed by transmission electron microscopy (TEM). A polyester membrane of $0.4\;{\mu}m$ pore size was determined as the most effective insert based on the change in the transepithelial electric resistance (TEER) value as well as the $^{14}C-mannitol$ transport study. The ALI method was effective in developing the tight junction as observed in the further increase in the TEER value and reduction in the permeability coefficient $(P_{app})$ of $^{14}C-mannitol$ transport. Results of the transport study of a model drug, budesonide, showed that the primary culture system developed in this study could be further developed and applied for in vitro nasal transport studies.

• Journal of Microbiology and Biotechnology
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• 제15권2호
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• pp.259-264
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• 2005

Transplantation of cultured chondrocytes can regenerate cartilage tissues in cartilage defects in humans. However, this method requires a long culture period to expand chondrocytes to a large number of cells for transplantation. In addition, chondrocytes may dedifferentiate during long-term culture. These problems can potentially be overcome by the use of undifferentiated or partially developed cartilage precursor cells derived from neonatal cartilage, which, unlike chondrocytes from adult cartilage, have the capacity for rapid in vitro cell expansion and may retain their differentiated phenotype during long-term culture. The purpose of this study was to compare the cell growth rate and phenotypic modulation during in vitro culture between adult chondrocytes and neonatal chondrocytes, and to demonstrate the feasibility of regenerating cartilage tissues in vivo by transplantation of neonatal chondrocytes expanded in vitro and seeded onto polymer scaffolds. When cultured in vitro, chondrocytes isolated from neonatal (immediately postpartum, 2 h of age) rats exhibited much higher growth rate than chondrocytes isolated from adult rats. After 5 days of culture, more neonatal chondrocytes were in the differentiated state than adult chondrocytes. Cultured neonatal chondrocytes were seeded onto biodegradable polymer scaffolds and transplanted into athymic mice's subcutaneous sites. Four weeks after implantation, neonatal chondrocyte-seeded scaffolds formed white cartilaginous tissues. Histological analysis of the implants with hematoxylin and eosin showed mature and well-formed cartilage. Alcian blue/ safranin-O staining and Masson's trichrome staining indicated the presence of highly sulfated glycosarninoglycans and collagen, respectively, both of which are the major extracellular matrices of cartilage. Immunohistochemical analysis showed that the collagen was mainly type II, the major collagen type in cartilage. These results showed that neonatal chondrocytes have potential to be a cell source for cartilage tissue engineering.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2003년도 생물공학의 동향(XIII)
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• pp.113-117
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• 2003

모낭은 복잡한 상호작용을 통하여 성장 및 주기가 조절되며 다양한 세포들로 구성진 기관이다. 본 연구에서는 조직공학적 방법을 이용하여 모낭을 구성하는 세포들을 분리 및 배양하였고 세포들의 형태와 증식양상을 관찰할 수 있었고, 기관배양의 방법을 통하여 in vitro에서 첨가물에 대한 모발의 성장 및 주기의 변화를 살펴보았다.