• 한국동물학회지
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• 제34권2호
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• pp.209-216
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• 1991

신경세포의 분화에 미치는 insulin의 영향을 알아보기 위하여 계배의 단뇌 신경아세포를 serum-free defined medium에서 배양하였다. Immunofluorescence실험을 통하여 신경특이단백질인 MAP-2는 신경아세포의 세포체와 신경돌기에 존재하는 것으로 나타났다. 또한, 배양액내에 insulin의 농도를 증가하면 신경아세포의 신경돌기 형성이 증가할 뿐 아니라 MAP-2의 합성도 증가하였다. 따라서, 신경아세포의 형태적분화와 생화학적 분화는 서로 밀접하게 연관되어 일어나는 것으로 추측되며, insulin은 신경특이단백질의 합성을 촉진시킴으로써 신경아세포가 신경돌기를 형성하며 분화되어 가는데 결정적인 역할을 하는 것으로 보인다.

• Archives of Pharmacal Research
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• 제20권4호
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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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• 제14권1호
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• pp.17-20
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• 2001

The effect of chicken IGF-I on protein synthesis of chicken embryo myoblasts cultured in serum-free medium was examined. When myoblasts were expanded to approximate 20-30% of well, the medium was changed to the serum-free medium including 0, 2, 20, 200 or 2000 ng/ml of recombinant chicken IGF-I. The culture medium including 10% fetal calf serum (FCS) was used as positive control. After 1 day of incubation, protein synthesis was measured by the incorporation of [$^3H$]-L-leucine. Thereafter cells were continued to incubate for further 18 hours, and the radioactivity in the protein was measured as an index of protein synthesis. The values for protein synthesis cultured in the serum-free medium without chicken IGF-I or with 2000 ng/ml of chicken IGF-I were the lowest. Protein synthesis was elevated with increasing chicken IGF-I concentration from 0 to 20 ng/ml. The values for protein synthesis in the 20 ng/ml and 200 ng/ml IGF-I groups were about half of that of the FCS group. The present study revealed that the potency of chicken IGF-I at the levels of 20 to 200 ng/ml to stimulate myoblast protein synthesis was about half of that of 10% FCS.

• KSBB Journal
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• 제5권1호
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• pp.87-94
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• 1990

본 논문에서는 10% fetal bovine serum(FBS)를 첨가한 RPMI 1640배지에 비하여 간염바이러스 표면항원에 대한 단일클론항체 생산에 있어서 효과적은 무혈청 배지에 낮은 온도의 혈청을 첨가하여 하이브리도마_2_c3.1 세포에서 그 효과를 조사하였다, 세포성장관 단일클론항체 생산을 증가시키기 위하여 기본 무혈청배지와 RPMI 1640 배지성분들의 농도를 균형있게 강화시킨 배지를 2:1(v / v)로 혼합하여 농축배지를 조성하고, 이 배지에 2mg / ml 인혈청 알부민 $5\;{\mu\textrm{g}}\;/\;ml$ insulin, $5\;{\mu\textrm{g}}\;/\;ml$ tran-sferrin, $10\;{\mu\textrm{g}}\;/\;ml$ monoethanolmine 등의 몇가지 무혈청 첨가물들을 첨가하였다. 이 농축 배지에 fetal bovine serum(FBS)과 supplemented bovine calf serum(sBCS)을 첨가하였을때의 세포성장과 단일클론항체 생산을 비교하여 FBS농도를 변화하여 세포성장과 단일클론항체생산의 증가를 시도하였다. 0.5%를 농축배지에 첨가함으로써 세포성장과 단일클론항체생산의 증가를 보았다. 최대 세포농도는 $3.06{\times}10^6$ cells / ml이었으며, 이때 생산된 단일클론항체는 10% FBS배지의 $43.0\;{\mu\textrm{g}}\;/\;ml$ 무혈청배지의 $50\;{\mu\textrm{g}}\;/\;ml$보다 3배이상 높은 $159.7\;{\mu\textrm{g}}\;/\;ml$이 생산되었다.

• Biotechnology and Bioprocess Engineering:BBE
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• 제11권4호
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• pp.319-324
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• 2006

Although the sera used in animal cell culture media provide the macromolecules, nutrients, hormones, and growth factors necessary to support cell growth, it could also be an obstacle to the production of recombinant proteins in animal cell culture systems used in many sectors of the biotechnology industry. For this reason, many research groups, including our laboratory, have been trying to develop serum-free media (SFM) or serum-supplemented media (SSM) for special or multi-purpose cell lines. The Chinese hamster ovary (CHO) cell, for example, is frequently used to produce proteins and is especially valuable in the large-scale production of pharmaceutically important proteins, yet information about its genome is lacking. Also, SFMs have only been evaluated by comparing growth patterns for cells grown in SFMs with those grown in SSM or by measuring the titer of the target protein obtained from cells grown in each type of medium. These are not reliable methods of obtaining the type of information needed to determine whether an SFM should be replaced with an SSM. We carried out a cDNA microarray analysis to evaluate MED-3, an SFM developed in our laboratory, as a CHO culture medium When CHO cells were cultured in MED-3 instead of an SSM, several genes associated with cell growth were down-regulated, although this change diminished over time. We found that the insulin-like growth factor (IGF) gene was representative of the proteins that were down-regulated in cells cultured in MED-3. When several key supplements - including insulin, transferrin, ethanolamine, and selenium - were removed from MED-3, the IGF expression was consistently down- regulated and cell growth decreased proportionately. Based on these results, we concluded that when an SFM is used as a culture medium, it is important to supplement it with substances that can help the cells maintain a high level of IGF expression. The data presented in this study, therefore, might provide useful information for the design and development of SFM or SSM, as well as for the design of genome-based studies of CHO cells to determine how they can be used optimally for protein production in pharmaceutical and biomedical research.

• 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 Nutrition and Health
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• 제25권2호
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• pp.109-115
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• 1992

This study was undertaken to determine the effect of dietary fat to carbohydrate ratio on plasma glucose. free fatty acid level and hepatic glucokinase activity in normal or insulin treated diabetic rats. Sprague-Dawley male rats were fed with 3 different but isocaloric diets for 5 weeks. Diet 1 made to have low fat(4% corn oil and 65.8% corn starch wt/wt) : diet 2 medium fat (12% : 47.8%) : diet 3 high fat (20% : 29.8%) In the normal rats an apparent increase of GK activity was observed from the animal fed low fat diet when compared with other groups. GK activities were decreased in all the alloxan-diabetic rats than the normal rats. When insulin was injected the GK activities in all the livers of alloxan-diabetic rats restored to normal level and GK activity was highest in low fat group. In the entire group significant relationships were seen between the plasma glucose and GK activities(r=-0.6, p<0.001) FFA levels and GK activities(r=-0.63 p<0.001) Both in normal and insulin treated diabetic rats significantly depressed level of hepatic GK activity was observed in the livers of animals fed high fat diet for 5 weeks and depressed level of GK activity may be related to insulin resistance.

• 한국미생물·생명공학회지
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• 제17권5호
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• pp.481-488
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• 1989

최근 단일클론항체의 상업적 이응가치가 증대하면서 그 수요량 또한 급격히 증가, 이를 뒷받침할 수 있는 대량생산 기술이 요구되고 있다. 본 연구에서는 쥐 하이브리도마 Alps 25-3의 대량배양을 위해 우선적으로 무혈청 배지 (serum-free media)의 개발을 시도하였다. 동물세포 배양액 IMDM과 Ham's F-12를 1:1로 혼합한 배지를 기본으로하여 ITES 용액 (insulin 10$\mu\textrm{g}$/$m\ell$, transferrin 10$\mu\textrm{g}$/$m\ell$, ethanolamine 10$\mu$M and selenium 30nM)을 첨 가하여 일차적으로 결정한 EBM(enriched basal medium)에 steroid hormones, vltamins, lipid, mineral 및 여러성분들을 각각 첨가하여 혈청과 대치될 수 있는 성분들의 최적농도를 조사하였다. 각 성분들의 개별효과와 함께 그들을 혼합하였을 때의 상호효과를 통해 혈청배지와 거의 동일한 성장을 보이는 무혈청배지 KM3(EBM with BSA 100$\mu\textrm{g}$/$m\ell$, mineral solution and 0.05% PEG)를 결정할 수 있었으며 이 배지에서의 최고 항체 생성량은 혈청배지의 약 80% 로 나타났다. 또한 무혈청 배지에서 여러 cell line의 성장과 그들의 항체 생성을 조사함으로써 다른 쥐 하이브리도마 세포배양에도 적응시킬 수 있음을 확인할 수 있었다.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2003년도 생물공학의 동향(XIII)
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• pp.171-173
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• 2003

본 연구에서는 생분해성 고분자 나노입자를 이용하여 부착성 세포의 세포군집 (cell aggregates) 형성을 촉진시켜 무혈청 배지에서 3차원적 부유 배양하는 방법을 개발했다. 생분해성 고분자 나노입자의 사용은 무혈청 배지 부유배양에서 부착성 동물세포인 HEK 293 세포의 세포군집 형성과 세포증식(나노입자를 사용하지 않은 대조군과 비교하여 2배 이상)을 촉진하였다. 일반적으로 무혈청배지 부유배양에 세포를 적응(adaptation)시키는 데에는 시간이 오래 걸리고 많은 비용이 드는데, 이 연구에서 개발된 방법은 이러한 세포적응 공정이 필요없다. 이 배양법은 여러 부착성 동물세포의 산업적 대량배양에 유용하게 사용될 수 있을 것이다.

• 한국수산과학회지
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• 제39권1호
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• pp.23-26
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• 2006

Effects of sera from several fish species and insulin on the development of cultured Silurus asotus hepatocytes were investigated. Hepatocytes with high viability (95%) were obtained from the livers of male catfish by two step collagenase perfusion. Isolated hepatocytes, initially showed a typical round-shape, firmly attached to the culture dish within 24 h. In the presence of catfish serum, hepatocytes attached each other, spread well on the dish and developed into monolayer after 3-4 days of incubation. Cells within the established monolayer became polygonal in shape and their nuclei and boundaries being clearly visible under the microscope. In contrast, when incubated in FBS-supplemented or serum-free medium, cells managed to form small clusters, each made of 2-10 cells. Cells in FBS-supplemented medium further developed into larger clusters. However, these clusters failed to develope into monolayer. In addition, when insulin was deprived from culture medium, formation of monolayer also failed. From these data, it can be concluded that the presence of both catfish serum and insulin is necessary for the formation of monolayer of catfish hepatocytes and the functional role of fish serum may differ from that of insulin and can not be displaced by FBS-supplementation.