• Clinical and Experimental Reproductive Medicine
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• v.19 no.2
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• pp.133-141
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• 1992

We examined effects of co-culture with human oviduct epithelial cells (HOEC) on the development of mouse and human embryos from early embryonic· stage to late morula or blastocyst stage (LM or B). In human, embryos were transferred and pregnancy rate was investigated. The HOEC, collected from surgically removed fallopian tube, were cultured in medium-199 supplemented with 20 % fetal cord serum (FCS). The HOEC were characterized by using immunocytochemical staining with anticytokeratin antibody and then used for cultures of mouse and human embryos. Results obtained from co-culture system were as follows. Development rate of mouse embryos was improved by co-culture system at late developmental stage (p<0.025). Human supernumerary embryos remained after transfer, unsuitable for freezing because of their poor quality, were co-cultured for 72hrs. Co-culture (78.79%) or conditioned medium (78.26%) system improved the developmemt rate, significantly, in comparision with control (11.11%)(p<0.00l). Co-cultured (85.71%) human zygotes for 24hrs showed the better development rate in comparision with control (50.00%) (p<0.01). When we transferred embryos cultured with the HOEC to patients, we obtained one pregnancy. Co-cultured human zygotes for 24hrs showed the better quality and viability for the replacement in comparision with control (p<0.01). In addition, improved pregnancy rate was obtained. Our results suggest that the co-culture system can rescue early degenerating embryos by improving early development and yield a resonable number of blastocyst for the appropriate replacement. The effect provided by cultured HOEC is not species specific for the development of embryos and it can be used to overcome in vitro blocks for the development. And also the co-culture system offers the possibility to freeze embryos at blastocyst stage which is more sucessful stage for the freezing. The HOEC monolayer may provide some stimulus via specific factor, which is unknown, to the development of embryos. Our results showed that the co-culture system with HOEC can be an alternative to conventional culture system.

• Journal of Veterinary Clinics
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• v.23 no.4
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• pp.411-415
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• 2006

Ascorbic acid has been used widely as a medium supplement to stimulate cell proliferation, but its effects on cell proliferation have not yet been elucidated, and no reports have analyzed effects on subcultured chondrocytes. Subcultured canine chondrocytes of passage one, two and four were cultured in monolayer and alginate beads with and without ascorbic acid. Cell proliferation was examined by 2,3-Bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide inner salt) (XTT) colormetric assay. Ascorbic acid stimulated cell proliferation significantly in both culture methods (p<0.05). The increased cell numbers by stimulation with ascorbic acid were significantly high in passage one cells compared to that of other passages. Differences in cell proliferative capacity by subculturing were not determined. These results suggest that ascorbic acid stimulated the proliferation of subcultured canine chondrocytes and enhanced it more in low-passage cells than in the other cells tested.

• Korean Journal of Animal Reproduction
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• v.18 no.1
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• pp.39-46
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• 1994

This experiment was carried out to develop an in vitro culture system for rabbit embryos. The zygotes or 2-cell embryos were collected from the oviducts of the superovulated and mated does with D-PBS/10% FCS at 24 hours after hCG injection. The in vitro developmental rate of blastocyst formation and the number of nuclei in the embryos were examined under the following treatments; 1) TCM-199 with 10% FCS, 2) EBSS with 10% FCS, 3) rabbit vitreous humor(VH), 4) TCM-199 with 10% FCS＋BOEC, 5) TCM-199 with 10% FCS＋ROEC, 6) EBSS with 10% FCS＋BOEC and 7) EBSS with 10% FCS＋ROEC. For a comparative study of in vivo and in vitro development, the fresh blastocysts, which were developed in vivo for 96 hours after hCG injection, were collected from the uterus and their numbers of nuclei were counted. 1. The zygotes or 2-cell embryos developed to the blastocyst stage in TCM-199, EBSS and VH at the rates of 93, 92 and 89%, respectively. 2. The higher developmental rates 95~98% of blastocyst formation was achieved when the embryos were co-cultured with a monolayer of bovine or rabbit oviductal epithelial cells in TCM-199 or EBSS. No significant difference in developmental rates was shown between bovine and rabbit oviductal epithelial cells. 3. In a comparative study of in vivo and in vitro development, the total numbers of nuclei were significantly less in the in vitro cultured embryos(104~224) than the in vivo developed embryos(1, 0090 at 96 hours after hCG injectin. 4. The mean cell cycle numbers in the embryos cultured for 72 hours in TCM-199 with 10% FCS, EBSS with 10% FCS, TCM-199 with 10% FCS＋BOEC, TCM-199 with 10% FCS＋ROEC, EBSS with 10% FCS＋BOEC and in vivo was 7.38, 6.63, 7.76, 7.69, 7.01 and 9.92, respectively. From these results, it can be suggested the optimal culture system for in vitro culture of rabbit embryos is a co-culture system with bovine or rabbit oviductal epithelial cells in TCM-199 with 10% FCS. Considering the significant reduction in total numbers of nuclei in the in vitro cultured embryos, the advanced research on development of in vitro culture system for rabbit embryos is expected.

• Clinical and Experimental Reproductive Medicine
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• v.24 no.2
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• pp.233-239
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• 1997

Embryos of most mammalian species grown in vitro would undergo developmental arrest at the approximate time of genomic activation. Stage-specific cell block and the resulting rapid loss of embryo viability in conventional culture media have limited the duration for which embryos may be cultured prior to transfer. As a result, embryos are usually transferred to the uterus at the 4-to 8-cell stage to avoid the loss of viability associated with long-term in vitro culture. Early transfer has led to asynchrony of the endometrium-trophectoderm interaction at the time of implantation and a resultant reduction in the rate of implantation. To overcome these problems, a variety of co-culture systems has been devised in which embryos can develop for a longer period prior to embryo transfer. Vero cells, derived from African green monkey kidney, share a common embryologic origin with cells from the genital tract. In addition, they are potentially safe to use, since they are highly controlled for viruses and other contaminants. Therefore, co-culture using Vero cells has been widely utilized to enhance embryo viability and development, although not without controversies. We thus designed a series of experiments to demonstrate whether Vero cells do indeed enhance mouse embryo development as well as to compare the efficacy of co-culturing mouse 1-cell embryos on Vero cell monolayer in both Ham's F-10 and human tubal fluid (HTF) culture media. 1-cell stage ICR mouse embryos were cultured either in the presence of Vero cells (Group A) or in conventional culture medium alone (Group B). In Ham's F-10 significantly more 3-to-8cell embryos developed in group A than group B (59.8 versus 10.0%; p<0.01). In contrast, there was no significant difference in embryonic development both group A and group B in HTF. However, significant differences were noted only in later embryonic stage (13 and 0%; p<0.05 of group A and B respectively, hatching or hatched). In Ham's F-10, we also could observe the beneficial effect of Vero cell on hatching process (70.7 and 42.1%; p<0.05 of group A and group B respectively).

• Journal of Embryo Transfer
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• v.12 no.2
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• pp.161-169
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• 1997

This study was conducted to compare the insemination time of bovine oocytes and determine the effects of glucose(1.5 mM) on the development of bovine embryos at early cleavage stage. Oocytes were matured for 24 h, followed by exposure to sperm and cultured in modified Tyrode's media drops or with bovine oviduct epithelial cell monolayer prepared in TCM199(BOECM). Insemination time and culture system were varied in each experiment. In experiment 1, to investigate the developmental capacity of bovine embryos after different time of exposure to sperm, bovine ova and sperm were co-incubated for 18, 30 or 54 h, respectively. The development to blastocysts of 30 and 54 h insemination groups were significantly higher(P<0.05) than 18 h group, and in case of blastocysts of cleaved embryos, 30 h group were significantly higher(P<0.05) than other groups. In experiment 2, we investigated the effect of glucose on early bovine embryos. After 18 h insemination, in vitro fertilized oocytes were separated following 3 groups ; G+0, C+24 and C+48. Oocytes of G+0 group were cultured in glucose added Tyrode's medium after fertilization, oocytes in C+24 and C+48 groups were cultured in glucose free Tyrode's medium after fertilization. After 24 h culture, G+24 group was moved to glucose added medium. All oocytes of 3 groups were moved to BOECM after 48 h culture. The rates of cleavage and development to blastocysts in G+0 group were significantly lower than other groups. In experiment 3, we determined the effects of glucose exposure from 8 to 20 h after insemination on the cleavage and development of oocytes. The oocytes in glucose added group had high capacity of cleavage and further development. This study shows that in bovine oocytes, the optimal exposure to sperm is 30 h and glucose exposure to bovine one-cell embryos is detrimental to their first cleavage and further development in vitro but there has no evidence of detrimental effect of glucose(1.5 mM) exposure to bovine embryos over the two-cell stage in vitro.

• Archives of Pharmacal Research
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• v.20 no.4
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• pp.297-305
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• 1997

A new serum-free defined medium was developed that supports the growth of normal rat mammary epithelial cells. Mammary organoids from the glands of female F344 rats were cultured in a serum-free medium. Monolayer culture colonies developed within a week and remained viable for months in culture. Upon subculture of one-week-old primary colonies, almost the same morphology of colonies was developed. The scrape loading/dye transfer technique showed that most of colonies that developed in a serum-free medium containing EGF, human transferrin, insulin, and hydrocortisone (basal serum-free medium, BSFM) failed to show cell-cell communication. However, colonies cultured in BSFM supplemented with prolactin, $E_2$, and progesterone (complete hormone serum-free medium, CHSFM) showed cell-cell communication at 14 days of primary culture or of subculture. By flow cytometry with FITCPNA and PE-anti-Thy-1.1 monoclonal antibody, we distinguished four RMEC subpopulations in cultures in both media: Thy-1.1+ cells, PNA+ cells, cells negative to both reagents and cells positive to both reagents. It is likely that combined prolactin, cortisol, and insulin in CHSFM stimulate terminal differentiation of clonogenic cells.

• Asian-Australasian Journal of Animal Sciences
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• v.8 no.4
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• pp.317-320
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• 1995

The effect of co-culture with cumulus cells and granulosa cells during maturation and development on in vitro developmental potency of follicular oocytes was examined. TCM-199 supplemented with 15% FCS and hormones was used as maturation medium. Sperm from frozen semen was capacitated in modified mTALP medium containing 0.3% BSA, $10{\mu}g/ml$ heparin and 5 mM/ml caffeine. The fertilized embryos were co-cultured on monolayer of cumulus cells or granulosa cells in TCM-199. The embryo co-cultured with cumulus cells showed higher percentage of embryo developed to morula and blastocyst (73.3%) than the embryo co-cultured without cumulus cells (30.8%). The percentage of oocytes developed to morula and blastocyst among cleaved oocytes was significantly (p < 0.05) higher in the oocytes co-cultured with cumulus cells during development (62.4%) than in the oocytes co-cultured with granulosa cells during maturation and with cumulus cells during development (52.3%), and in the oocytes co-cultured with granulose cells during development (52.8%). The results of this study indicate that co-culture of in vitro fertilized embryos with cumulus cells in the development medium increased the rate of embryos developed to morula and blastocyst among cleaved oocytes.

• The Journal of the Convergence on Culture Technology
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• v.8 no.1
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• pp.371-377
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• 2022

This study examines whether there are subjects and learners to pay attention to as 'processes' that have not been dealt with in Korean vocabulary education due to prioritizing learning outcomes, educational outcomes, and objects. In addition, the purpose of this study was to examine the educational value of the neologism and to suggest data construction method for it. Proposal to create a 'single-level list' of neologisms as a preliminary work to create a dictionary as a learning material to teach new words to academic purpose learners, taking neologism as the vocabulary in the blind spot and foreign academic purpose learners as learners in the blind spot stage. did The 'single-layered list' is to divide new words by period into coined words, meanings, culture, etc. and construct them as data. Through this study, we will help systematically teach Korean vocabulary by adding vocabulary to be learned as a 'process' to the results of Korean vocabulary education so far.

• Proceedings of the Korean Society of Developmental Biology Conference
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• 2003.10a
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• pp.108-108
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• 2003

The purpose of this is to investigate the effects of vitrification in open pulled straws (OPS) on in vitro survival of porcine embryos. Blastocysts were produced by in vitro fertilization of slaughterhouse-derived, in vitro matured oocytes with frozen-thawed boar semen, and subsequent culture on granulosa cell monolayer. After frozen-thawing, embryos were culture in NCSU-23 medium with 5 mM hypotaurine, 4 mg/$m\ell$ BSA and 10 ng/$m\ell$ for 48 hrs to survival tests. When blastocysts were frozen-thawed by OPS methods, the embryos with normal morphology were 32.1, 34.5 and 38.9 % in early blastocyst, blastocyst and expanded blastocyat stages. The rates of partial damaged embryos were significantly (P<0.05) higher in early biastocysts than expanded blastocysts. In another experiment, the embryos frozen by OPS methods were cultured for 48 hrs for survival and developmental rates in vitro. The proportions of embryos hatched were 11.8, 20.2 and 33.3% in embryos frozen-thawed at stages of early blastocyst, blastocyst and expanded embryos. On the other hand, The proportions of embryo with normal morphology after culture were 23.5, 25.0 and 33.3% in embryos frozen-thawed at stages of early blastocyst, blastocyst and expanded embryos. These finding indicate the possible broader application for OPS methods that this procedure described is relatively harmless, that it can be used for blastocysts of different developmental stages.

• KSBB Journal
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• v.13 no.3
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• pp.231-237
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• 1998

An attenuated Japanese encephalitis virus (JEV) clone SA-14-14-2(Vero) was obtained through successive adaptation of a primary cell adapted strain, SA-14-14-2(PDK) to Vero cell, a continuous line of monkey kidney cells. The virus titer reached above the 107 plaque forming unit (pfu) per mL of culture supernatant with 3 passages in Vero cells and was maintained close to this level in the further passages. The optimum temperature for the virus growth was $35^{\circ}C$. Growth pattern of the virus indicated that optimum time for the virus harvest is 4 days post infection and the virus accomplished rapid initial propagation even in medium containing no serum supplement. The roller bottle (RB) system and the spinner flask (SF) system using micro-carrier (Cytodex-1) for the JEV cultivation were explored. When RB, SF, and T-flask culture system were compared, there was no significant difference in virus yield. Furthermore, the results indicated that virus could be harvested multiple times from 3 days to 9 days post infection; neither severe cytopathic effect (CPE) in the infected cells nor the decrease in the titer was observed on duration of 9 days.