• Journal of Periodontal and Implant Science
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• v.30 no.1
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• pp.39-52
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• 2000

Polypeptide growth factor belong to a class of potent biologic mediator which regulate cell differentiation, proliferation, migration and metabolism. 1,25-dihydroxyvitamin $D_3$ decrease cell proliferation, and stimulate alkaline phosphatase activity which express in osteoblast during cell differentiation period. IGF-I is known to stimulate cell proliferation and differentiation too. 1,25-dihydroxyvitamin $D_3$ is known to increase IGF-I binding sites and IGF binding protein which inhibite the effect of IGF. The purpose of this study is to evaluate potential role of IGF-I as mediator that control the action of 1,25-dihydroxyvitamin $D_3$. MC3T3-E1 cell were seeded $5{\times}10^5/ml$ at 100mm culture plate in ${\alpha}-MEM$ containing 10% fetal bovine serum. After 48 hour incubation period, medium were changed ${\alpha}-MEM$ containing 5% fetal bovine serum. After 24 hours, $10^{-9}M$ 1,25-dihydroxyvitamin $D_3$ added. Total mRNA was extracted at 0, 6, 24, 48, 72 hour. PRPCR method was programed for the detection of IGF-I mRNA. In the both groups of 1,25-dihydroxy vitamin $D_3$ treated and control, alternative splicing form of IGF-I, IGF-IA and IGF-IB were expressed. In the 1,25-dihydroxyvitamin $D_3$ treated group, IGF-I mRNA expression was matained until 24 hour, there after expression was decresed. MC3T3-E1 cell were seeded $2.5{\times}10^4/ml$ at 24well plate in ${\alpha}-MEM$ containing 10% fetal bovine serum. After 48 hour incubation period, medium were changed ${\alpha}-MEM$ containing 3% fetal bovine serum. After 24 hours, $10^{-9}M$ 1,25-dihydroxyvitamin $D_3$ and 10 ng/ml IGF-I were added separately or together. Cell were cultured for 1 and 3 days, $2{\mu}Ci/ml\;[^3H]$ -thymidine was added for the last 24h of culture of each days. ${[^3H]}$-thymidine incorporation in to DNA was measured and expressed counter per minute(CPM). DNA synthetic activity was significantly decreased by 1,25-dihydroxyvitamin $D_3$ both at 1 day and 3 day, and in the combination group of 1,25-dihydroxyvitamin $D_3$ and IGF-I, DNA synthetic activity was also decreased both at 1 day and 3 days. IGF-I did not affect the DNA synthetic activity compared to control group both at 1 day and 3 day. From the above results, 1,25-dihydroxyvitamin $D_3$ was potent inhibitor of cell proliferaton in MC3T3-E1 cells. It assumed that the effect of 1,25-dihydroxyvitamin $D_3$ on osteoblast proliferation may be mediated in part by decreased level of IGF-I.

• Journal of Veterinary Clinics
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• v.7 no.2
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• pp.517-519
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• 1990

In vitro maturation and fertilization of oocytes collected from slaughtered bovine ovaries were investigated. Immature bovine extrafollicular oocytes were cultured for 24 hrs. in TCM 199 supplemented with fetal calf serum in a humidified CO$_2$ incubator. Fertilization in vitro was performed using frozen-awed bull semen which was treated by Ca Ionophore A23187. Fourty percentage of oocytes cultured had matured to the metaphase II ; There were-no effects of the concentration of fetal calf serum and of the addition of HEPES on the maturation rate. The mean proportions of in vitro fertilized eggs and of cleaved eggs were 23.1％ and 14.4％, respectively.

• International Journal of Oral Biology
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• v.35 no.1
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• pp.1-5
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• 2010

Human fibroblasts that maintain the structural integrity of connective tissues by secreting precursors of the extracellular matrix are typically cultured with serum. However, there are potential disadvantages of the use of serum including unnatural interactions between the cells and the potential for exposure to animal pathogens. To prevent the possible influence of serum on fibroblast cultures, we devised a serum-free growth method and present in vitro data that demonstrate its suitability for growing porcine fetal fibroblasts. These cells were grown under four different culture conditions: no serum (negative control), 10% fetal bovine serum (FBS, positive control), 10% knockout serum replacement (KSR) and 20% KSR in the medium. The proliferation rates and viabilities of the cells were investigated by counting the number of cells and trypan blue staining, respectively. The 10% FBS group showed the largest increase in the total number of cells ($1.09\;{\times}\;10^5\;cells/ml$). In terms of the rate of viable cells, the results from the KSR supplementation groups (20% KSR:64.7%; 10% KSR: 80.6%) were similar to those from the 10% FBS group (68.5%). Moreover, supplementation with either 10% ($3.0\;{\times}\;10^4\;cells/ml$) or 20% KSR ($4.8\;{\times}\;10^4\;cells/ml$) produced similar cell growth rates. In conclusion, although KSR supplementation produces a lower cell proliferation rate than FBS, this growth condition is more effective for obtaining an appropriate number of viable porcine fetal fibroblasts in culture. Using KSR in fibroblast culture medium is thus a viable alternative to FBS.

• 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.

• Mass Spectrometry Letters
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• v.5 no.1
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• pp.30-33
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• 2014

A solid phase extraction (SPE) method was optimized for the quantitative analysis of perfluorooctanoic acid (PFOA) in serum using hydrophilic-lipophilic balance SPE and LC-MS/MS. Fetal bovine serums spiked with $^{13}C_8$-PFOA before or after SPE were used as test samples for evaluation of the SPE efficiency. Simultaneous evaluation of matrix effects and absolute SPE recovery for $^{13}C_8$-PFOA in serum using different sample pre-treatments and SPE conditions allowed optimization of SPE process efficiency with minimal matrix effect and decent SPE recovery. Introduction of protein precipitation as a sample pre-treatment procedure for serum samples before SPE generally decreased matrix effect in LC-MS/MS analysis and provided more stable recovery of PFOA.

• Clinical and Experimental Reproductive Medicine
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• v.16 no.2
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• pp.161-171
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• 1989

The development of 2-cell mouse embryos to the blastocyst stage in vitro has been used as quality control for the culture media and supplements employed for human in vitro fertilization and embryo transfer(IVF-ET). 2-cell mouse embryos were cultured to the blastocyst stage in SECM, Medium 199-Earle's, Ham's F-10 I , Ham's F-10 II , Hoppe & Pitts, MEM and $HT_6$. The protein supplements contained in media were bovine serum albumine, fetal bovine serum and human fetal cord serum. The results were as follows; 1. The successful development was 81.3% in Medium 199-Earle’s, 91.9% in Ham’s F-10 I and 97.1% in $HT_6$. 2. 2-cell mouse embryos developed properly in all supplements but the best development was particularly noted in $HT_6$ media when HFCS was supplied as protein supplement.

• Archives of Plastic Surgery
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• v.32 no.4
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• pp.529-532
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• 2005

Expanding cells ex-vivo is very important in tissue-engineering. Culture medium is usually supplemented with fetal bovine serum(FBS) in most of the experiments. However, cells grown in bovine serum media may posses the possibilities of disseminating bovine diseases and/or stimulating the patient's immune reactions. To overcome these problems, autologous or homologous serum should be used instead of the FBS. The purpose of this study is to compare cell proliferation and collagen synthesis depending on the kind of sera mixed on media and to provide a guideline on applying established experimental data to clinical cases. Human dermal fibroblasts were obtained from four patients. Five thousand cells per well in 96-well plates were incubated DMEM/F-12 Nutrient with varying serum mixture; 10% autologous serum, 10% homologous serum, and 10% FBS. Five days after incubation fibroblast proliferation and collagen production were determined by MTT assay and CICP enzyme immunoassay. The mean cell number were; $3.95{\times}10^4/well$, $2.97{\times}10^4/well$ and $2.30{\times}10^4/well$, respectively. The average amounts of collagen synthesized were; 238.13 ng/ml, 204.88 ng/ml, and 163.88 ng/ml in each. These results show that the use of human serum mixture may contribute to, not only preventing disseminated infection of bovine diseases. but also increase cell proliferation and collagen synthesis without simulating the patient's immune reactions.

• Journal of Embryo Transfer
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• v.6 no.1
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• pp.33-40
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• 1991

In vitro developmental ability of early preimplantation monse embryos was shown to be depend on the embryonic stages, media and snpplements and their interaction(Experiment 1). The development of I-cell embryos were more promoted in the complex medinm(Ham's Fl0) than in the simple one(m-KRB), but that of 2-cell embryos showed the reverse effect. The bovine serum albumin(BSA) as a medium snpplement more promoted the development of I- and 2-cell embryos, compared with human fetal cord serum(HCS). On the other hand, the harmful effect of HCS was especially shown on the early cleavage in the embryonic development of the two stages. The effect of serum, in the respect of interaction between media and snpplements. was also more significantly appeared in m-KRB than Ham's Fl0. In the experiment 2, when the harmful effect of HCS was compared with that of fetal bovine serum(FBS), the former more promoted the development of l - and 2-cell embryos than the latter. The effect of HCS was more significantly shown in the development of I-cell than that of 2-cell embryos. Conclusively, as I- and 2-cell embryos were different in the requirements for the in vitro development. the optimal medium and supplement have to be selected for each embryonic stage. It is also respected to the better result if it take into consideration into the kinds of sera when serum is used for culture of early preimplantation embryos.

• Korean Journal of Animal Reproduction
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• v.20 no.2
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• pp.191-196
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• 1996

The objective of this study were to investigate the effects of culture media and additives on the development of bovine in vitro matured(IVM) and in vitro fertilized(IVF) oocytes. In experiment 1, bovine oocytes were cultured in droplets of TC 199 supplemented with 10% fetal bovine serum(FBS) with or without hormones (5$\mu\textrm{g}$/ml FSH, 5$\mu\textrm{g}$/ml LH, 1$\mu\textrm{g}$/ml E2). Cleavage rates of embryos cultured for 40~44hrs after IVF were higher when embryos were cultured in TC 199 supplemented hormones (68.1%, 921/35) than without hormones (52.7%, 77/146), but the percentages of development beyond morulae stage were not difference (20.7%, 19.4%). In experiment 2, the effects of various media such as TC 199, synthetic oviduct fluid(SOF), CR1aa with different energy source (fatal bovine serum, FBS; bovine serum albumin, BSA) on developmental capacity of IVM/IVF bovine embryos were investigated. The developmental rates into morulae and blastocysts were 27.1, 10.7, 6.3 and 0%, respecitvely, in CR1aa plus 3mg/ml BSA, SOF plus 10% FBS, TC 199 plus 10% FBS, SOF plus 3mg/ml BSA. In experiment 3, the comparisons of bovine embryos developed to morulae and blastocysts in different culture media (TC 199, SOF, CR1aa, Menezo's B2) were investigated. The developmental capacity beyond morulae stage were 32.9, 26.6, 11.1 and 7.1%, respectively, in Menezo's B2 plus BSA, CR1aa plus BSA, SOF plus BSA, TC 199 plus FBS medium. The cell numbers of the blastocyst were not different in different cultrue media. In experiment 4, bovine embryos were co-cultured with vobine oviduct epithelial cells(BOEC) in TC 199 plus FBS, SOF plus BSA, CR1aa plus BSA, Menezo's B2 plus BSA. The morula and blastocyst rates were 44.7, 32.9, 26.0 and 23.3%, respectively, in CR1aa TC 199, SOF, and Menezo's B2 medium. The cell numbers of the blastocyst were similar to those of blastocyst developed in different culture media.

• The Journal of Internal Korean Medicine
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• v.21 no.3
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• pp.433-441
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• 2000

Objective : To analyze the effects of Yukmijihwang-tang, Taeksa-tang, Silbi-um on mesangial cell proliferation, fibronectin synthesis and MHC-class II expression. Methods : Laboratory studies were performed with the method of surface enzyme immunoassays or flow cytometry after addition of peripheral blood mononuclear cells(PBMC) supernatants treated with medications using the cultured human mesangial cells. Results : 1. Silbi-um produces more suppressive effect than control group and hydrocortisone group on the mesangial cell proliferation. In Yukmijihwang-tang, Taeksa-tang and Silbi-um, mesangial cell proliferation significantly decreased than in hydrocortisone group 2. In the 'without fetal bovine serum' study, Yukmijihwang-tang take more suppressive effect than Control group on the fibronectin synthesis. In the 'with fetal bovine serum' study, Yukmijihwang-tang, Taeksa-tang, Silbi-um all have suppressive effect, but it hasn' t any statistical significance. 3. Yukmijihwang-tang, Taeksa-tang, Silbi-um all have a suppressive effect on the MHC-class II expression. Conclusions : Herb medicine generally show a suppressive effect on the suppression of the mesangial cell proliferation, fibronectin synthesis and MHC-class II expression.