• Proceedings of the Korean Society of Developmental Biology Conference
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• 2001.08a
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• pp.38-38
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• 2001

TM4 Sertoli cell의 체외 관강형성 유도에 미치는 세포외기질 (ECM) 및 hepatocyte growth factor (HGF)의 역할과 세포분화 과정에서 MMP의 발현의 변화를 조사하였다. Matrigel bed(60%, v/v) 상에서 배양한 TM4 cell은 무혈청 조건하에서 chain 분화단계를 거쳐 cord의 구조로 분화하였다. 그러나 이후의 분화는 일어나지 않았다. TM4 cell에서 c-MET (HGF receptor)의 발현을 확인하였으며 HGF를 첨가한 배양액에서 분화가 촉진되었으며, cord에서 tubule로의 분화가 유도되었다. 또한 TM4 cell의 분화는 MMP-2 및 MMP-9의 발현이 증가를 수반하였으며 HGF는 MMPs의 발현을 증가시켰다. GFR-Matrigel과 성장인자인 HGF는 무혈청 배지에서 TM4 cell의 체외에서 관강형성에 필요한 환경을 제공하며, MMP-2 및 -9은 TM4 cell의 체외분화 과정에서 조절역할을 수행하는 것으로 사료된다.

• Microbiology and Biotechnology Letters
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• v.17 no.5
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• pp.481-488
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• 1989

A serum-free medium that could be used for the large-scale culture of mouse hybridoma to produce monoclonal antibodies was developed. The medium was based on a 1:1 mixture of Iscove's Modified Dulbecco's Medium and Ham's F-12, supplemented with insulin 10$\mu\textrm{g}$/$m\ell$, transferrin 10$\mu\textrm{g}$/$m\ell$, ethanolamine 10$\mu$M and selenium 30nM (designated EBM (enriched basal medium) with the supplements). The effect of various supplements of steroid hormones, vitamins, lipid and mineral salts was investigated and their optimal concentration was determined to replace fetal calf serum (PCS). These components were added respectively and then added by way of two or three combination to discern of which component combination was effective to the culture of hybridoma. As a result, serum-free medium KM3 (EBM with BSA 100$\mu\textrm{g}$/$m\ell$, mineral cocktail and 0.05% PEG) was deter-mined. The hybridoma Alps 25-3 cultured in this medium showed almost the same growth rate as in medium added with 2% fetal bovine serum. However, the antibody concentration from KM3 cultures was 80% of that obtained from culture with FCS. KM3 was also examined for the culture of other mouse hybridomas, KW, A4W & HCGK, and it was confirmed that it could support the growth of these hybridomas and the production of monoclonal antibodies.

• 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.

• Korean Journal of Animal Reproduction
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• v.24 no.3
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• pp.269-279
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• 2000

The present study was carried out to evaluate the effect of glycosaminoglycans added to the culture medium on the mouse embryo development to the blastocyst stage. In vitro fertilized mouse oocytes were cultured in Ham's F-10 supplemented with 10% FBS either in the absence or presence of 0.1, 0.5, 1.0 mg/$m\ell$ hyaluronic acid, chondroitin sulfate, and dermatan sulfate, respectively. After 4 days in culture, embryos developed to blastocysts were observed in all groups. There was a significant increase in blastocyst yield in the presence of hyaluronic acid and chondroitin sulfate (p<0.05), whereas dermatan sulfate was ineffective. Development to the blastocyst stage was best supported in 0.1, 0.5, 1.0 mg/$m\ell$ hyaluronic acid and 0.5mg/$m\ell$ chondroitin sulfate. It is concluded that hyaluronic acid and chondroitin sulfate support the development of mouse oocyte fertilized in vitro to the blastocyst stage. Furthermore, these results suggest that glycosaminoglycans can be utilized to support embryo development in vitro as a nutrient instead of serum.

• KSBB Journal
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• v.21 no.4
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• pp.306-311
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• 2006

Cell preservation is indispensable in animal cell culture process and should be established according to the cell characteristics. In this study, we experimented hypothermic preservation of CHO cells that is widely used in pharmaceutical industry to produce therapeutic proteins and established a stable method of preservation. The highest viability of CHO cells was obtained when the cells were preserved using rolling tube, which means the cells should be suspended to avoid the cell lumping during the preservation. Also, we obtained superior preservation result under the anaerobic condition. To evaluate the serum-free media as a preservation solution, we investigated cell growth after hypothermic preservation using serum-free media. High cell viability and normal cell growth was observed during 10 days using serum-free media. Moreover, we found that more effective preservation when ${\alpha}$-tocopherol and retinoic acid is added to media as an additive. In the case of 1 liter large scale hypothermic preservation using established protocol, cell viability and growth rate was obtained as good as small scale one. This study is considered to be helpful for hypothermic preservation of CHO cells and large scale hypothermic preservation may be available through the further studies.

• Journal of Life Science
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• v.34 no.3
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• pp.170-178
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• 2024

Adipose-derived stem cells (ADSCs) are capable of differentiation into multiple lineages of cells, which has attracted attention for clinical therapy. However, ADSCs have poor proliferation capacity and a short life span in culture, which is an impediment in the application to clinical use. Previously, to overcome growth disadvantages, we had established an immortalized ADSC line (ADSC-T) by introducing the SV40 T antigen coding gene into primary human ADSC. In the present study, we evaluated the differentiation potential of this cell line and assessed the anti-inflammatory effect of its conditioned medium (CM). ADSC-T appeared to maintain the differentiation potential into adipocyte and chondrocyte. The CM of ADSC-T suppressed the NF-κB activity and its target gene expression of COX-2 and iNOS. Furthermore, the phosphorylations of MAPKs, including ERK, JNK and p38, were suppressed by the ADSC-T CM. The expressions of pro-inflammatory cytokines such as TGF-β, TNF-α, IL-6, and IL-13 were also suppressed by the CM of ADSC-T. In the Nc/Nga atopic model mice, the CM showed therapeutic effect on DNCB-induced atopic dermatitis. These results indicate that the immortalized ADSC-T maintains the beneficial properties of primary ADSC and could be a versatile cell source for not only research into ADSC but also for production of CM suitable for clinical application.

• Journal of Embryo Transfer
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• v.20 no.1
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• pp.71-77
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• 2005

To investigate the influence of relaxin and insulin on the ovarian steroid secretion of porcine granulosa cells, we used porcine granulosa cells partially luteinized in a primary culture and examined the production of progesterone and $17{\beta}-estradiol$. Porcine granulosa cells were cultured in the presence of serum for 48 h after attachment and subsequently in the absence of serum fur 24 h. To confirm the dose dependency of relaxin or insulin, various concentrations (10, 100, 1000 ng/ml) of relaxin or insulin were added in the medium for the last 24 h, respectively. To investigate the combinational effect of relaxin and insulin, 100 ng/ml relaxin and/or 100 ng/ml insulin were added in the medium for the last 24 h in the presence or absence of luteinizing hormone (100 ng/ml). The medium was collected and used for radioimmunoassay to measure the production of progesterone and $17{\beta}-estradiol$. Relaxin or insulin increased the production of progesterone by dose dependency, respectively while they had no effect of the production of $17{\beta}-estradiol$. Relaxin (100 ng/ml) and/or insulin (100 ng/ml) significantly increased the production of progesterone in the presence of luteinizing hormone while they had no effect of the production of $17{\beta}-estradiol$. In conclusion, relaxin and/or insulin increased the progesterone secretion of porcine granulosa-lutein cells in vitro while had no effect on the production of $17{\beta}-estradiol$ and had no synergism on the effects. The effects of relaxin and/or insulin on the production of progesterone were augmented by the presence of luteinizing hormone.

• Journal of Embryo Transfer
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• v.22 no.2
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• pp.137-141
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• 2007

Antioxidants were well known to be essential supplements in the complex media and serve as a reservoir of oxygen. In this study, Hanwoo COCs (cumulus oocytes complexes) were matured and developed in L-cysteine-TCM199 and analyzed metaphase chromosome. Maturation rate of Hanwoo COCs were 73.4%, 94.6% in 0.1% PVA, 0.1 mM L-cysteine, respectively and showed significantly different between the treatments (p<0.05). Blastocyst formation were revealed 20.3%, 10.0% in 5% FBS+TCM199, 0.1 mM L-cysteine+1% BSA, respectively. There were no significant difference among treatment groups. Metaphase chromosome were showed 18.3%, 12.0% in 5% FBS-TCM199, 0.1 mM L-cysteine, respectively and analyzable chromosome were 6.1%, 4.0% and had no differences between the treated groups. In the case of in vitro developmental stages, metaphase chromosome were showed 18.3%, 12.0% in $4{\sim}16$ cells stage, 43.1%, 13.0% in morulae stage and 94.8%, 100.0% in blastocyst stage. These results suggested L-cysteine has beneficial role for in virto maturation and development in Hanwoo COCs.

• Journal of Embryo Transfer
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• v.13 no.3
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• pp.235-243
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• 1998

In this study, we investigated the effects of three kinds of culture medium (Charles and Rosenkrans; CRlaa, Tyrode's; TALP, synthetic oviduct fluid: SOF), insulin transferrin + selenium complex (ITS), macromolecules(polyvinyl alcohol: PVA, fetalb-ovine serum: FBS) and NaCl on the development of early bovine embryos. In experiment 1, there were no differences in embryo development among three kinds of embryo culture medium (CR $l_{aa}$ , TALP, SOF). In experiment 2, BSA, FBS and PVA were added each in TALP as macromolecule sources. The developmental rates of embryos in BSA or FBS added TALP were significantly higher than in PVA added one (p〈0.01), but there was no difference between BSA and FBS added groups. In experiment 3, bovine embryos were cultured in TALP with the following supplements: BSA alone(1, 3 or 8 mg/ml, each) or BSA(1, 3 or 8 mg/ml, each)+ITS (10$\mu\textrm{g}$/m1 insulin, 5 $\mu\textrm{g}$/ml transferrin, 5 ng/ml selenium). In higher concentration of BSA and ITS supplemented groups, the developmental rates over compacted morula were higher than others, but there was a significant effect of ITS only in 1 mg/ml of BSA added group (p〈0.05). In experiment 4, the effect of reduced concentration of NaCl was evaluated. The developmental rate over compacted morula in the medium containing 90 mM of NaCl was higher than in 114 mM group (p〈0.05). In conclusion, BSA could be used as a macromolecule source in bovine embryo culture, and ITS, as a serum substitute, could be used for improving of embryonic development. Also, reduction of NaCl concentration from 114 mM to 90 mM may improve the development of bovine embryos.bryos.

• Journal of Animal Science and Technology
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• v.48 no.6
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• pp.805-812
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• 2006

The purpose of this study was to investigate the effect of Matrigel on the development of bovine embryos after in vitro fertilization. Bovine embryos were cultured in Ⅰ) SOF+ 0.8% BSA(SOF-B), Ⅱ) SOF+ 0.8% BSA plus 0.8% Matrigel(SOF-M) and III) SOF+0.8% BSA and 10% FBS(SOF-BF). The addition of Matrigel appeared not to increase the proportion of blastocysts (SOF-B, 26.6%; SOF-M, 28.2%; SOF-BF, 26.2%). However, the proportion of hatched blastocysts were significantly increased(P<0.05) by Matrigel(SOF-B, 23.7%; SOF-M, 48.7%; SOF-BF, 18.5%). The means of cell number blastocyst was not significantly different among the treatment groups(SOF-B, 172.7±35.5; SOF-M, 175.1±37.4; SOF-BF, 172.8±38.1). The proportion of apoptotic cells in blastocyst was also found to be not significant among the treatment groups(SOF-B, 3.6±3.2%; SOF-M, 4.3±2.6%; SOF-BF, 4.9±4.3%). In this experiment, Matrigel appeared to support embryonic hatching of bovine embryos. Results suggest that Matrigel, as extracellular matrix components, may be another avenue for formulating more physiological culture system in serum-free culture.