• Clinical and Experimental Reproductive Medicine
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• 제31권4호
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• pp.209-215
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• 2004

Objective: Embryonic stem (ES) cells could be differentiated into the specific cell types by alternation of culture condition and modification of gene expression. This study was performed to evaluate the differentiation protocol for mouse and human ES cells to insulin secreting cells. Methods: Undifferentiated mouse (JH-I) and human (Miz-hESI) ES cells were cultured on STO feeder layer, and embryoid bodies (EBs) were formed by suspension culture. For the differentiation, EBs were cultured by sequential system with three stage protocol. The differentiating ES cells were collected and marker gene expressions were analyzed by seIni-quantitative RT-PCR in each stage. Amount of secreted insulin levels in culture media of human ES cells were measured by human insulin specific RIA kit. Results: During the differentiation process of human ES cells, GATA-4, a-fetoprotein, glucose transporter-2 and Ngn-3 expression were increased whereas OctA was decreased progressively. Insulin and albuInin mRNAs were expressed from stage IT in mouse ES cells and from stage III in human ES cells. We detected 3.0~7.9 IlU/rnl secretion of insulin from differentiated human ES cells by in vitro culture for 36 days. Conclusion: The sequential culture system could induce the differentiation of mouse and human ES cells into insulin secreting cells. This is the fIrst report of differentiation of human ES cells into insulin secreting cells by in vitro culture with serum and insulin free medium.

• Journal of Microbiology and Biotechnology
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• 제13권6호
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• pp.926-936
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• 2003

Overexpression of human Bcl-2 protein in recombinant Chinese hamster ovary (rCHO) cells producing humanized antibody (SH2-0.32) considerably suppressed sodium butyrate (NaBu)-induced apoptosis during batch culture by using commercially available serum-free medium, which extended the culture longevity. Due to the extended culture longevity provided by the anti-apoptotic effect of Bcl-2 overexpression, the final antibody concentration of 14C6-bcl-2 culture (Bcl-2 high producer, $23\;\mu\textrm{g}\;ml^{-1}$) was 2 times higher than that of the $SH2-0.32-{\Delta}bcl-2$ culture (cells transfected with bcl-2-deficient plasmid, $10.5\;\mu\textrm{g}\;ml^{-1}$) in the presence of NaBu. To determine the effect of NaBu/Bcl-2 overexpression on the molecular integrity of protein products, antibodies purified from 14C6-bcl-2 and $SH2-0.32-{\Delta}bcl-2$ cultures in the presence of NaBu were characterized by using various molecular assay systems. For comparison, antibody purified from the parental rCHO cell culture (SH2-0.32) in the absence of NaBu was also characterized. No significant changes in molecular weight of antibodies could be observed by SDS-PAGE. From GlycoSep-N column analysis, it was found that the core oligosaccharide structure ($GlcNAc_2Man_3GlcNAc_2$) was not affected by NaBu/Bcl-2 overexpression, while the microheterogeneity of N-linked oligosaccharide structure was slightly affected. Compared with the antibody produced in the absence of NaBu, the proportion of neutral oligosaccharides was increased from 10% (14C6-bcl-2) to 16% ($SH2-0.32-{\Delta}bcl-2$) in the presence of NaBu, which was accompanied by the reduced proportion of acidic oligosaccharides, especially of monosialylated and disialylated forms. The changes in microheterogeneous oligoformal structures of antibody in turn affected the mobility of antibody isoforms in isoelectric focusing (IEF), resulting in the occurrence of some more basic antibody isoforms produced in the presence of NaBu. However, the antigen-antibody binding properties were not changed by alteration of glycosylation pattern. The competitive enzyme-linked immunosorbent assay (ELISA) showed that the antibody produced by NaBu/Bcl-2 overexpression maintained its antigen-antibody binding properties with binding affinity of about $2.5{\times}10^9{\;}M^{-1}$. Taken together, no significant effects of NaBu/Bcl-2 overexpression on the molecular integrity of antibodies, produced by using serum-free medium, could be observed by the molecular assay systems.

• 한국식품영양과학회지
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• 제32권3호
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• pp.437-443
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• 2003

선행연구에서 라이코펜이 HT-29세포의 증식을 억제하는 것을 관찰하였기 때문에 본 연구는 그 기전을 연구하기 위하여 수행되 었다. 라이코펜이 HT-29 세포의 사멸을 유도하는지 조사하기 위해서 여러 농도의 라이코펜이 포함된 배지에서 세포를 4일 동안 배양하였다. 라이코펜 농도의 증가에 따라 사멸되는 세포에서 나타나는 특징의 하나인 DNA fragmentation이 증가하는 것을 관찰하였다. Western blot을 수행하여 얻은 결과에 의하면 라이코펜이 IGF-IR, IRS-1, PI3K, Akt와 같은 IGF-IR pathway에 속하는 단백질의 수준을 감소시켰다. IGF-IR의 인산화를 유도하기 위해서 라이코펜이 포함된 배지에서 세포를 배양하고 IGF-I을 첨가하여 0, 5, 10, 60분간 배양한 다음 IGF-IR antibody를 이용하여 immunoprecipitation을 수행하였다. 라이코펜은 IGF-I에 의한 IGF-IR, IRS-1의 인산화와 IGF-IR와 PI3K의 결합을 감소하고 인산화된 Akt를 감소시켰다. 이와 같은 IGF-IR signaling의 억제는 이 대장암세포에 존재하는 IGF-II의 autocrine loop을 억제하는 원인이 될 수 있어, 라이코펜의 암세포증식을 억제하는 기전 중의 하나가 될 수 있다. 라이코펜은 토마토와 그 가공품에 많이 존재하는 물질로 자연적인 식사를 통해 많이 섭취할 수 있는 물질이다. 라이코펜의 항암 기전을 밝혀냄으로써 미래 암예방과 치료를 위한 중요한 기능성 영양소를 생산할 수 있는 기초를 마련해줄 수 있을 것으로 기대된다.

• International Journal of Oral Biology
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• 제33권3호
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• pp.113-116
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• 2008

Cementum is the mineralized tissue of the tooth. It is similar to bone in several aspects but it differs from bone. Human bone marrow stromal cells (BMSC) and human cementum derived cells (HCDC) (10,000 $cells/cm^2$) were plated in 6 well plates as feeder cells. The next day, mouse bone marrow cells (1.5 million $cells/cm^2$) were added. One group of these plates were incubated in serum-free conditioned medium (SFCM) generated from BMSC or HCDC supplemented with 2% FBS, parathyroid hormone (PTH), 1, 25 dihydroxyvitamin $D_3$ (Vit. $D_3$) and dexamethasone, or plain medium with the same supplements. Another group of plates were cocultured with BMSC or HCDC in plain medium supplemented with 2% FBS, PTH, Vit. $D_3$ and dexamethasone. Plates grown without SFCM or coculture were used as controls. After 10 days, the cells were stained for tartrate-resistant acid phosphatase (TRAP). BMSC were found to support osteoclast formation under normal conditions. This was inhibited however by both SFCM generated from HCDC and also by coculture with HCDC. In addition, HCDC themselves did not support osteoclast formation under any conditions. Our results thus indicate that HCDC do not support osteoclast formation in vitro and that soluble factor (s) from HCDC may inhibit this process. In addition, we show that this inhibition also involves an active mechanism that is independent of osteoprotegerin, a feature that may distinguish cementoblasts from other cells present in periodontium.

• Reproductive and Developmental Biology
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• 제33권3호
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• pp.171-177
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• 2009

Spermatogonial stem cells(SSCs) only are responsible for the generation of progeny and for the transmission of genetic information to the next generation in male. Other in vitro studies have cultured SSCs for proliferation, differentiation, and genetic modification in mouse and rat. Currently, information regarding in vitro culture of porcine Germline Stem Cell(GSC) such as gonocyte or SSC is limited and is in need of further studies. Therefore, in this study, we report development of a successful culture system for gonocytes of neonatal porcine testes. Testis cells were extracted from $10{\sim}14$-day-old pigs. These cells were harvested using enzymatic digestion, and the harvested cells were purified with combination of percoll, laminin, and gelatin selection techniques. The most effective culture system of porcine gonocytes was established through trial experiments which made a comparison between different feeder cells, medium, serum concentrations, temperatures, and $O_2$ tensions. Taken together, the optimal condition was established using C166 or Mouse Embryonic Fibroblast(MEF) feeder cell, Rat Serum Free Medium(RSFM), 0% serum concentration, $37^{\circ}C$ temperature, and $O_2$ 20% tension. Although we discovered the optimal culture condition for proliferation of porcine gonocytes, the gonocyte colonies ceased to expand after one month. These results suggest inadequate acquirement of ingredients essential for long term culture of porcine GSCs. Consequently, further study should be conducted to establish a successful long-term culture system for porcine GSCs by introducing various growth factors or nutrients.

• Journal of Periodontal and Implant Science
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• 제27권4호
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• pp.669-684
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• 1997

Polypeptide growth factors belong to a class of potent biologic mediators which regulate cell differentiation, proliferation, migration and metabolism. IGF-I is polypeptides secreted by skeletal cells and is considered as regulators of bone formation. The purpose of this study is to evaluate the effects of IGF-I on bone nodule formation and alkaline phosphatase activity of MC3T3-E1 cells. MC3T3-E1 cells were seeded at $1{\times}10^4$ cells/well, $1{\times}10^5$ cells/well in alpha-modified Eagle medium containing 10% fetal bovine serum, 10 mM ${\beta}-glycerophosphate$ and $5O{\mu}g/ml$ of ascorbic acid. Before 48 hours of indicated time, medium were changed with serum free medium. After 24 hours, 0.1, 1, 10 ng/ml IGF-I were added to the cells and cultured for 3, 7, 14, 21, 28 days. And histochemical analysis was done and ALP activity was measured and was expressed as nmol/min/mg of protein. The bone nodule formation in MC3T3-E1 cells of IGF-I was seen at 21, 28 days, but there were no difference between control group and experimental groups. The ALP activity decreased when it is compare to control 2 group except for 1 ng/ml, 10 ng/ml IGF-I of 21-day-groups and 1 ng/ml IGF-I of 28-day-groups. Dose response effects of IGF-I of ALP activity in MC3T3-E1 cells were seen the highest ALP activity at 1ng/ml until 21days and the highest ALP activity at 10 ng/ml of 28 daygroups. The peak times were seen at 7-day group, 14-day group on control group and experimental group respectively, and 1 ng/ml group was the highest ALP activity, From the above results, IGF-I was not seen notable effect on bone nodule formation and decreased ALP activity of MC3T3-E1 cells but the use of IGF-I to mediate biological stimulation of MC3T3-E1 cells shows promise for future therapeutic application.

• 대한수의학회지
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• 제31권2호
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• pp.179-187
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• 1991

These studies were carried out to investigate the effects of the size of follicles, semen types, capacitation methods, and additions of hormones, estrous cow serum(ECS), fetal calf serum(FCS), bovine follicular fluid(BFF) and matured cumulus cell(MCC) to the medium on in vitro maturation and fertilization of bovine follicular oocytes. The ovaries were obtained from slaughtered Korean native cows. The follicular oocytes surrounded with cumulus cells were recovered by aspirating follicular fluid from the visible follicles of diameter 3~5mm. The follicular oocytes were cultured in TCM-199 medium containing hormones, FCS, ECS, BFF and MCC for 24~48hrs. in an incubator with 5% $CO_2$ in air at $38.5^{\circ}C$ and then matured oocytes were again cultured for 18~20 hrs. with motile capacitated spermatozoas the TCF (Tyrode calcium free) solution containing $100{\mu}g/ml$ of heparin. The results obtained in these experiments were summarized as follows: 1. The oocytes classified as "A,B,C,D and Degenerative" depending morphological integrity and those 61.4%, 12.1%, 19.2%, 4.2% and 3.0% of the total oocytes recovered, respectively. The maturation and fertilization rate of the A, B, C class follicular oocytes, cultured in the TCM-199 medium supplemented with 10% FCS were 89.1%, 78.0%, 52.6% and 78.1%, 66.1, 33.3%, respectively. 2. The average number of the follicular oocytes recovered from follicles size, 1~2mm, 3~5mm and above 5mm in diameter were 67, 98 and 63, respectively. The maturation and fertilization rate of the follicular oocytes, cultured in the TCM-199 medium were 56.7%, 82.5%, 46.0% and 44.8%, 71.4%, 28.6%, respectively. 3. The fertilization and cleavage rate of the follicular oocytes, inseminated with spermatozoas of epididymal cauda, neat and frozen semen were 63.3%, 73.3%, 70.0% and 32.7%, 37.8% 38.3%, respectively. 4. The fertilization and cleavage rate of follicular oocytes, fertilized with capacitated spermatozoas by heparin, BFF and HIS methods were 70.0%, 53.8%, 34.2% and 38.3%, 23.1%. 17.1%, respectively. And the fertilization and cleavage rate were higher method of heparin. 5. The maturation and fertilization rate of follicular oocytes, cultured in the TCM-199 medium supplemented with 5%, 10%, 15%, 20% and FSH, HCG, 17, $\beta$-estradiol were 76.0~82.3% and 26.2~70.0%, and those values were higher the supplementation than non-supplementation. 6. The maturation and fertilization rate of the follicular oocytes cultured in TCM-199 medium supplemented with 5~20% ECS and FCS were 74.0~80.6%, 26.2~30.0% and 71.7~76.9%, 51.9~58.0%, and the values were higher the supplement of ECS than FCS. 7. The maturation rate (68.0~64.6%) and fertilization rate(59.6~60.4%) of follicular oocytes cultured in TCM-199 medium supplemented with 10% FCS and 20~30% BFF were higher than those of follicular oocytes cultured TCM-199 medium supplemented with 10% FCS and 10% and 50% BFF. 8. The maturation rate(76.5%) and fertilization rate (61.7%) of follicular oocytes cultured in TCM-199 medium supplemented with 10% FCS and $1{\times}10^6/ml$ MCC were higher than those of follicular oocytes cultured in TCM-199 medium supplemented with 10% FCS and $1{\times}10^{{4}{\sim}{5}}/ml$ and $1{\times}10^8/ml$ cumulus cells.

• Journal of Veterinary Science
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• 제24권1호
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• pp.4.1-4.16
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• 2023

Background: In vitro culture of preantral follicles is a promising technology for fertility preservation. Objectives: This study aims to investigate an optimized three-dimensional (3D) fetal bovine serum (FBS)-free preantral follicle culture system having a simple and easy operation. Methods: The isolated follicles from mouse ovaries were randomly divided in an ultra-low attachment 96-well plates supplement with FBS or bovine serum albumin (BSA) culture or encapsulated with an alginate supplement with FBS or BSA culture. Meanwhile, estradiol (E₂) concentration was assessed through enzyme-linked immunosorbent assay of culture supernatants. The diameter of follicular growth was measured, and the lumen of the follicle was photographed. Spindle microtubules of oocytes were detected via immunofluorescence. The ability of oocytes to fertilize was assessed using in vitro fertilization. Results: The diameters were larger for the growing secondary follicles cultured in ultra-low attachment 96-well plates than in the alginate gel on days 6, 8, and 10 (p < 0.05). Meanwhile, the E2 concentration in the BSA-supplemented medium was significantly higher in the alginate gel than in the other three groups on days 6 and 8 (p < 0.05), and the oocytes in the FBS-free system could complete meiosis and fertilization in vitro. Conclusions: The present study furnishes insights into the mature oocytes obtained from the 3D culture of the preantral follicle by using ultra-low attachment 96-well plate with an FBS-free system in vitro and supports the clinical practices to achieve competent, mature oocytes for in vitro fertilization.

• 한국가축번식학회지
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• 제17권2호
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• pp.85-92
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• 1993

In the previous study, we observed that Purine has a time dependent effect in maintaining the oocytes in meiotic arrest, and human fetal cord serum(HFCS) and human mature follicular fluid(HMFF) reverse the GVBD suppressed by purines. And it was reported that purine has a harmful effect on the development of oocytes or embryos, when they were cultured for a long time, in vitro. Therefore this study was performed to investigate the effects of purine on extrusion rates of 1st pb and viability of oocytes cultured for a long time, in vitro. Immature oocytes(GV stage) were collected from ovaries of 25~28 day old ICR mice at 48 hrs after PMSG injection. Cumulus-enclosed and denuded oocytes collected were assigned randomly to one of several culture conditions. Some of the oocytes were cultured in 4mM hypoxanthine for 24hr, and the extrusion rates of 1st pb and viability of the oocytes were assessed at every 12 hrs. In the viability, the oocytes showed granulation, pigmentation of cytoplasm or lysis of 1st pb extruded were regarded as degenerating oocytes. Also some of the oocytes were cultured in hypoxanthine for 12 hrs then the resulting oocytes were transferred to hypoxanthine-free medium and cultured for 12 hrs to determine whether the inhibitory effect of hypoxanthine on the 1st pb extrusion was reversible. The rest of the oocytes were cultured in medium containing hypoxanthine and adenosine for 18 hrs to compare the 1st pb extrusion be attendant upon hte concentration of HFCS or HMFF supplemented. Hypoxanthine suppressed the extrusion of 1st pb and viability of the oocytes significantly, when they were cultured for more than 12 hrs and the harmful effect of hypoxanthine was showed in denuded oocytes, prominently. The suppressive effect of hypoxanthine was reversed by just removal of the hypoxanthine from the cultrue medium. Also there was no difference in reverse the pb extrusion rate suppressed between HFCS and HMFF. The extrusion rate of 1st pb in medium containing adenosine and hypoxanthine was increased in line with the concentration of HFCS or HMFF supplemented. Hypoxanthine suppressed the extrusion of 1st pb and viability of the oocytes significantly, when they were cultured for more than 12 hrs and the harmful effect of hypoxanthine was showed in denuded oocytes, prominetly. The suppressive effect of hypoxanthine was reversed by just removal of the hypoxanthine fromthe culture medium. Also there was no difference in reverse the pb extrusion rate suppressed between HFCS and HMFF. The extrusion rate of 1st pb in medium containing adenosine and hypoxanthine was increased in line with the concentration of HFCS or HMFF supplemented.

• Preventive Nutrition and Food Science
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• 제1권2호
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• pp.220-226
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• 1996

To investigate the effect of long chain n-3 polyunsaturated fatty acids on breast cancer cell growth, estrogen-dependent MCF-7 human breast cancer cells were cultured serum-free DMEM media containing 0.5$\mu\textrm{g}$/ml of differnet kinds of fatty acids; linoleic acid(LA), arachidonic acid(AA), eicosapentaenoic acid(EPA) and docosahexaenoic acid acid(DHA) and 1, 0.1, 0.2, 0.5and 1.0ng/ml 17$\beta$-estradiol as well as 10$\mu\textrm{g}$/mi insulin and 1.25 mg/ml delipidized bovine serum albumin for 3 days. Cell growth monitored by MTT assay was lower in DHA and EPA treatments as compared to LA treatment, but not with AA treatment. Estrogen concentrations at which cell growth was initially stimulated were 0.1ng/ml for LA and DHA treatments and 0.2ng/ml for EPA and AA treatments, but the degree of stimulation was 25~30% lower in DHA and EPA treatments than in LA treatment. Fatty acid analysis showed that each fatty acid in culture medium was well incoporated into celluar lipid. Protein kinase C activity of cells was most elevated in LA treatment from 2 to 8 hours of culture followed by DHA, EPA, and AA treatments. It is concluded that inhibitions of n-3 DHA and EPA on breast cancer cell growth as compard with n-6 LA is mediated via changes in membrane fatty acid composition reducing estrogen sensitivity and increasing protein kinase C activity.