• 한국가축번식학회지
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• 제17권2호
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• pp.75-83
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• 1993

Purine has been identified in the preparation of follicular fluid and shown an activity in maintaining oocyte meiotic arrest. Therefore this study was performed to examine the inhibitory effect of purine on germinal vesicle break down(GVBD) in the presence and absence of human fetal cord serum(HFCS) or human mature follicular fluid(HMFF), as a protein source, in vitro culture. Immature oocytes(GV stage) were collected from ovaries of 21∼28 days old ICR mice by puncturing the antral follicles with a fine needle, at 48 hrs after PMSG injection. Some of the oocytes were denuded by drawing the cumulus-enclosed(complex) oocytes in and out of a pasteur pipet. Complex oocytes and denuded oocytes were cultured 3 hrs. in T6 media containing 0.75mM adenosine or/and 4mM hypoxanthine, with HFCS or HMFF. Their GVBD rates were observed at every 1 hr. during the culture time. Both adenosine and hypoxanthine have shown a time-dependent inhibitory effect on GVBD in complex and denuded oocytes and the inhibitory effect was maximized in culture medium containing hypoxanthine and adenosine. HFCS and HMFF increased the GVBD rates in the presence of the purines, thus HFCS and HMFF may contain a factor that could reverse the inhibitory effect of purines. Also complex oocytes were more sensitive to not only the inhibitory effect of purines but the promoting action of HMFF on GVBD than denuded oocytes. Therefore it was reconfirmed that granulosa cells play an important part in meiotic arrest and resumption.

• 한국발생생물학회지:발생과생식
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• 제16권4호
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• pp.339-351
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• 2012

Fetal bovine serum (FBS) is the most frequently used serum for the cultivation of mammalian cells. However, since animal-derived materials might not be appropriate due to safety issues, allogeneic human serum (HS) has been used to replace FBS, particularly for the culture of human cells. While there has been a debate about the advantages of HS, its precise effect on human adult stem cells have not been clarified. The present study aimed to investigate the effect of HS on the human eyelid adipose stem cells (HEACs) in vitro. When HEACs were cultivated in a medium containing 10% HS, many cells moved into several spots and aggregated there. The phenomenon was observed as early as 9 days following 10% HS treatment, and 12 days following 5% HS plus 5% FBS treatment. However, the aggregation was never observed when the same cells were cultivated with 10% FBS or bovine serum albumin. To examine whether cell density might affect the aggregation, cells were seeded with different densities on 12-well dish. Until the beginning of aggregation, cells seeded at low densities exhibited the longest culture period of 16 days whereas cells seeded at high densities showed the shortest period of 9 days to form aggregation. The number of cells was $15.1{\pm}0.2{\times}10^4$ as the least for the low density group, and $29.3{\pm}2.8{\times}10^4$ as the greatest for the high density group. When human cord blood serum or normal bovine serum was examined for the same effect on HEACs, interestingly, cord blood serum induced the aggregation of cells whereas bovine serum treatment has never induced. When cells were cultivated with 10% HS for 9 days, they were obtained and analyzed by RT-PCR. Compared to FBS-cultivated HEACs, HS-cultivated HEACs did not express VIM, and less expressed GATA4, PALLD. On the other hand, HS-cultivated HEACs expressed MAP2 more than FBS-cultivated HEACs. In conclusion, human adult stem cells could move and form aggregates by the treatment with human body fluids.

• International Journal of Oral Biology
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• 제37권2호
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• pp.43-50
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• 2012

The use of high throughput screening (HTS) in drug development is principally for the selection new drug candidates or screening of chemical toxicants. This system minimizes the experimental environment and allows for the screening of candidates at the same time. Umbilical cord-derived stem cells have some of the characteristics of fetal stem cell and have several advantages such as the ease with which they can be obtained and lack of ethical issues. To establish a HTS system, optimized conditions that mimic typical cell culture conditions in a minimal space such as 96 well plates are needed for stem cell growth. We have thus established a novel HTS system using human umbilical cord derived-mesenchymal stem cells (hUC-MSCs). To determine the optimal cell number, hUC-MSCs were serially diluted and seeded at 750, 500, 200 and 100 cells per well on 96 well plates. The maintenance efficiencies of these dilutions were compared for 3, 7, 9, and 14 days. The fetal bovine serum (FBS) concentration (20, 10, 5 and 1%) and the cell numbers (750, 500 and 200 cells/well) were compared for 3, 5 and 7 days. In addition, we evaluated the optimal conditions for cell cycle block. These four independent optimization experiments were conducted using an MTT assay. In the results, the optimal conditions for a HTS system using hUC-MSCs were determined to be 300 cell/well cultured for 8 days with 1 or 5% FBS. In addition, we demonstrated that the optimal conditions for a cell cycle block in this culture system are 48 hours in the absence of FBS. In addition, we selected four types of novel small molecule candidates using our HTS system which demonstrates the feasibility if using hUC-MSCs for this type of screen. Moreover, the four candidate compounds can be tested for stem cell research application.

• 한국동물학회지
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• 제29권4호
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• pp.283-293
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• 1986

사람의 alpha-fetoprotein(AFP)에 대한 모노클론 항체의 생산 및 분석을 위하여, 태아조직을 재료로 추출법, DEAE-cellulose 및 concanavalin A-Sepharose, Cibacron blue F3GA-agarose, immunoadsorbent column등의 흡착크로마토그라피법에 의해 AFP를 분리하였다. 총 534g의 태아조직에서 AFP의 量은 8.76 mg으로서 순수분리되었음을 확인하였다.

• Korean Chemical Engineering Research
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• 제44권1호
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• pp.73-80
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• 2006

본 연구에서는 제대혈 유래의 조혈줄기세포를 효과적으로 배양하기 위한 선행 연구로서 세포 배양 환경에 따른 조혈줄기세포 증식능의 변화를 관찰하였다. 제대혈의 단핵구 세포에서 분리한 $CD34^+$ 세포를 성장인자 조성-I(이하, coc-I) (EPO, GM-CSF, SCF, IL-3) 및 성장인자 조성-II(이하, coc-II) (TPO, G-CSF, SCF, IL-6, Flt3/Flk-2 ligand)가 포함되어 있는 IMDM(Iscove's modified Dulbecco's medium) 및 무혈청 배지(serum free media, SFM)에서 배양하였으며, 우태아혈청(FBS)의 첨가 영향, 2차원 및 3차원 배양 후 각 조건에서의 세포 증식 및 콜로니 형성능을 비교하였다. 일반적으로 coc-I에서의 세포 증식 및 콜로니 증식이 coc-II에서보다 높았다. 3차원 배양(methocult)에서는 가장 높은 세포 증식($2,258{\pm}456$배)을 나타냈으며, 같은 조성의 2차원 배양(IMDM + coc-I + FBS)에서는 가장 높은 콜로니 증식(BFU-E: $652{\pm}19$, CFU-GM: $520{\pm}58$, CFU-GEMM: $339{\pm}100$배)이 나타났다. 배지를 기준으로 보면, coc-II 조성에 우태아혈청이 포함되지 않은 경우를 제외한 모든 경우에서 세포 증식 및 콜로니 증식이 무혈청 배지에서보다 IMDM에서 높았다. 결론적으로, 모든 배양 조건 중에서 'IMDM + coc-I + FBS' 및 'IMDM + coc-I'에서 가장 좋은 콜로니 증식을 보였으며, 우태아혈청의 첨가 및 2차원 배양 조건이 콜로니 증식에 더 효과적인 것으로 확인되었다. 본 연구 결과는 앞으로 조혈줄기세포의 체외 증식에 필요한 공정개발이나 생물반응기 설계에 유용한 정보를 제공할 수 있을 것으로 사료된다.

• Clinical and Experimental Reproductive Medicine
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• 제21권2호
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• pp.183-190
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• 1994

Purpose of the present study was to find the optimal culture conditions for the maturation and fertilization of immature oocytes by the use human body fluids and gonadotropins (Gn) in the mouse model. Cumulus-enclosed mouse immature oocytes were incubated in the medium containing various human body fluids with or without Gn in vitro, and examined to confirm nuclear maturation (NM) and fertilization. Female ICR mice were stimulated with 7.5 IU pregnant mares' serum gonadotropin (PMSG). Cumulus-enclosed immature oocytes were isolated at 48-52 hr post PMSG injection and cultured in TCM 199 supplemented with various concentrations (20, 50, and 70%) of human body fluids such as fetal cord serum (hCS), follicular fluid (hFF), peritoneal fluid (hPF) and amniotic fluid (hAF) in the presence or absence of 10 IU/ml PMSG and 10 IU/ml human chorionic gonadotropin (hCG) for 18 hr. Fetal calf serum (FCS) was used as a control for the supplements. Matured oocytes were fertilized with sperm collected from the epididymis of male mice. Fertilization was conducted in T6 medium containing 15 mgl ml bovine serum albumin, and confirmed at 6 hr post-insemination. Evaluation of nucler maturation and fertilization was carried out by rapid staining using fuchin. There was no significant difference between the effects of human body fluids and FCS supplements on nuclear maturation of cumulus enclosed mouse immature oocytes. When maturation medium was supplemented with 20% hPF or 20% hAF, fertilization rates were significantly (P<0.01) lower than that of 20% FCS, hCS and hFF groups. However, higher concentrations of body fluids during IVM were not more beneficial on fertilizability of oocytes. The addition of Gn significantly increased the fertilization rates in hPF and hAF groups (hPF without Gn; 51.5%, compared with 85.1% for addition of Gn, and hAF without Gn; 30.1% compared with 85.8% for addition of Gn) at 20% concentration. These results suggest that human body fluids at 20% concentration and gonadotropins can be used as supplements for the maturation of mouse immature oocytes in vitro. When gonadotropins supplemented with the human body fluids in the maturation medium, fertilizability of mouse immature oocytes was increased in hPF and hAF groups. These results can be applied to maturation of human immature oocytes in vitro.

• Clinical and Experimental Reproductive Medicine
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• 제19권2호
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• pp.125-131
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• 1992

The possible effect of human follicular fluid(hFF) on the growth and development of fertilized oocytes and embryos is important because the fallopian tubes are exposed to FF after follicular rupture and the processes of fertilization and embryo cleavage occur inside the fallopian tubes. Previously, it was suggested that human FF might adversely affect on the development of early mouse embryos. In order to investigate the effect of hFF on the development of embryos, early mouse embryos were cultured in media containing various protein sources as bovine serum albumin(BSA), fetal cord serum(FCS) and FF. And we evaluated the development of early mouse embryos in terms of the morphology, cleavage rate, and cell count of blastcysts. There were no significant differences in the morula and blstocyst formation rates of 2-cell mouse embryos cultured in the media containg three different protein sources and three different concentrations of FF. The blastocyst formation rate of 1-cell mouse embryo cultured in FF group was significantly higher than that cultured in BSA group(P<0.05). The morula and blastocyst formation rates of 2-cell mouse embryos of the group cultured in the media containing FF were comparable with those of other two groups, in addition, the cell count of blastocysts of FF group in the 2-cell embryo culture was higher than those of BSA group and HCS group(P<0.01), and this finding was also noted in 1-cell embryo culture. There was no difference in the morula and blastocyst formation rates of the 2-cell mouse embryos cultured in the media containing different concentrations of FF. These results suggest that mature human follicular fluid has no inhibitory activity on the development of early mouse embryos even in high concentration and may be a good protein source which is positively associated with the development of mouse embryos in vitro especially in 1 cell embryo culture.

• Clinical and Experimental Reproductive Medicine
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• 제20권2호
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• pp.165-176
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• 1993

These studies were carried out to investigate the optimal freezing protocol for 2 cell mouse embryos and to find the probability of quality control with 2-cell embryos frozen. The embryos showed the best survival by the protocol composed of a freezing solution with the cryoprotectants(1.5M propanediol + 0.1M sucrose), and a 2-steop thawing method(room temperature, 20 sec-37$^{\circ}C$, 20 sec). The developmental ability of frozen-thaw 2-cell embryos did not differ from that of fresh 2-cell embryos in m-KRB medium with 0.4% bovine serum albumin. But development of frozen-thaw embryos was depended on the supplements of the medium. In the albumin-free medium, the developmental rate(rate of blastocysts) was significantly reduced, compared with that in the medium with 0.4% BSA. Also, when frozen-thaw embryos were cultured in the meduim with human fetal cord serum(HCS), the developmental rate of frozen-thaw embryos was sligtly reduced, compared with that of fresh 2-cell embryos. Finally, frozen-thaw 2-cell mouse embryos were more sensitive to the toxic agent of disposable-plastic syringe. Therefore, toxicity of medium could be effectively detected by frozen-thaw 2-cell mouse embryos.

• Clinical and Experimental Reproductive Medicine
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• 제22권3호
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• pp.279-285
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• 1995

Despite the frequent incidence of embryo fragmentation in early human embryos, the reason of the embryo fragmentation has not been known yet. This study was conducted to investigate the histological difference(s) between fragmented (FR) and non-fragmented (NFR) human embryos focusing on comparison of mitochondrial distribution and protein synthesis. Multi-pronuclei zygotes (MPZ) such as three or more pronuclei containing in human in vitro fertilization and embryo transfer (IVF-ET) program were used for this study. MPZ were cultured in TCM-199 supplemented with 10% of human fetal cord serum (hFCS) in 5% $CO_2$ incubator at $37^{\circ}C$ for 24 hours. The cleaved embryos to 2-4 cells after 24 hours were grouped by their grade of fragmentation. Embryos were stained with Rhodamine123 (Rh123) and fluorescence was evaluated under the fluorescence microscope through PB 450-490 filter (Leitz). Regarding to protein synthesis during early human embryogenesis, there is no significant difference in the amount of synthetic proteins between FR and NFR embryos. Distribution of cytoplasmic organelles in embryos was evaluated by transmission electron microscope (TEM). The cytoplasmic distribution of mitochondria was different between FR and NFR embryos. The mitochondrial distribution was even in NFR, whereas severely aggregated in FR. It is not able to clarify in the present study whether this uneven mitochondrial distribution in FR embryo is the reason for embryo fragmentation or is the result from fragmentation. Physiological disparity related to the mitochondrial distribution may be one of the reasons for embryo fragmentation. Further studies should be addressed to investigate the physiological differences between FR and NFR embryos.

• Clinical and Experimental Reproductive Medicine
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• 제19권2호
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• pp.169-174
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• 1992

Previous studies have indicated that immunological factor is responsible for the infertility. We have detected sperm antibodies in ZIFT patients which grouped as fertilization failure (A; n=18) and low fertilization rate (${\leq}50%$)(B; n=20). Patients, however, had normal oocytes and sperms. We collected serum from wives and semen from husbands and donors (fertile sperm), if it was needed. We examined class, binding patterns and amounts of antisperm antibodies(ASA) by direct and indirect immunobead binding assay. In group A, 11 husbands were ASA positive showing 62.2% and 61.1% binding with IgA and IgG, respectively, and two wives were ASA positive showing 70.0% and 71.0% binding with IgA and IgG, respectively. Binding sites were mainly at the head of sperms (84%). In group B, 8 husbands were ASA positive showing 37.5% and 40.0% binding with IgA and IgG, respectively, and two wives were ASA positive showing 41.3% and 42.0% binding with IgA and IgG, respectively. Binding sites were also mainly at the head of sperms (78%). For the treatment of ZIFT patients who had fertilization failure at the first trial, we used albumin fractionation method and dilution method with 30% fetal cord serum (FCS) to reduce the titer of ASA. We used partial zona dissection (P.Z.D.) method for wives who have antisperm antibodies in their serum. According to represented method, we could inhance the fertilization rate to 60.0% by albumin fractionation and 20.0% by P.Z.D., respectively. We concluded that the use of micromanipulation like P.Z.D. or the other sperm processing methods is required to increase a chance of fertilization. This result suggested that it should be a prerequisite to test antisperm antibodies prior to entering assisted reproductive technologies (ART) programs.