• 한국가축번식학회지
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• 제18권4호
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• pp.299-307
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• 1995

전능성을 지닌 우 수정란 세포배양기술 체계 확립은 가축육종에 중요한 의의를 지닌다. 이러한 체계는 1) 핵치환에 의한 다수의 클론동물 생산에 대한 기전, 2) 형질전환세포를 선발하기 위한 marker의 사용으로 효과적인 유전자 전이체계와 3) site specific 유전자 전이에 대한 기전 또는 homologous DNA서열 재조합에 의한 결손 등에 대한 기전을 이해하는데 이용될 수 있다. 우 수정란세포의 배양은 배반포 내부 세포괴, 상실배와 16∼20세포기로부터 확립하였다. 이들 모든 세포들은 생쥐 배아간 세포형태와 유사하였으며 배양시 분화와 증식에서 다능성을 나타내었다. 배양체계는 미세소적이나 배양용기, 우 도는 설치류 섬유아 세포주를 단기간 배양 또는 장기간 배양방법을 이용하였다. 유사분열시 요구되는 배양체계나 배양액 그리고 분화 억제에 대한 괄목할만한 장점은 아직 밝혀지지 않고 있다. 현재 16∼20 세포기의 배양세포의 전능성에 대해서는 알려져 있지 않다. 배양된 ICM세포 전능성은 27일간 배양한 ICM 세포로부터 4마리의 산자 생산에 의해 입증되었다.

• 한국발생생물학회지:발생과생식
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• 제16권4호
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• pp.353-361
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• 2012

Human embryonic stem (ES) cells are a potential source of cells for developmental studies and for a variety of applications in transplantation therapies and drug discovery. However, human ES cells are difficult to culture and maintain at a large scale, which is one of the most serious obstacles in human ES cell research. Culture of human ES cells on MEF cells after disassociation with accutase has previously been demonstrated by other research groups. Here, we confirmed that human ES cells (H9) can maintain stem cell properties when the cells are passaged as single cells under a feeder-free culture condition. Accutase-dissociated human ES cells showed normal karyotype, stem cell marker expression, and morphology. We prepared frozen stocks during the culture period, thawed two of the human ES cell stocks, and analyzed the cells after culture with the same method. Although the cells revealed normal expression of stem cell marker genes, they had abnormal karyotypes. Therefore, we suggest that accutase-dissociated single cells can be usefully expanded in a feeder-free condition but chromosomal modification should be considered in the culture after freeze-thawing.

• Journal of Microbiology and Biotechnology
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• 제32권1호
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• pp.126-140
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• 2022

Stem cells can be applied usefully in basic research and clinical field due to their differentiation and self-renewal capacity. The aim of this study was to establish an effective novel therapeutic cellular source and create its molecular expression profile map to elucidate the possible therapeutic mechanism and signaling pathway. We successfully obtained a mesenchymal stem cell population from human embryonic stem cells (hESCs) cultured on chemically defined feeder-free conditions and treated with connective tissue growth factor (CTGF) and performed the expressive proteomic approach to elucidate the molecular basis. We further selected 12 differentially expressed proteins in CTGF-induced hESC-derived mesenchymal stem cells (C-hESC-MSCs), which were found to be involved in the metabolic process, immune response, cell signaling, and cell proliferation, as compared to bone marrow derived-MSCs(BM-MSCs). Moreover, these up-regulated proteins were potentially related to the Wnt/β-catenin pathway. These results suggest that C-hESC-MSCs are a highly proliferative cell population, which can interact with the Wnt/β-catenin signaling pathway; thus, due to the upregulated cell survival ability or downregulated apoptosis effects of C-hESC-MSCs, these can be used as an unlimited cellular source in the cell therapy field for a higher therapeutic potential. Overall, the study provided valuable insights into the molecular functioning of hESC derivatives as a valuable cellular source.

• 한국수정란이식학회지
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• 제26권2호
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• pp.111-115
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• 2011

In general, zona pellucida (ZP) of the blastocyst has to be removed first, then either isolated the inner cell mass (ICM) or ZP-removed whole blastocyst, which is then cultured on the feeder layer to induce ICM outgrowth for the generation of embryonic stem cells (ESC). However, it is unclear whether ICM isolation before seeding on feeder layer is beneficial or not because the interaction between ICM and trophoblasts may affect cellular growth and/or pluripotency during the culture on the feeder. In the present study, two ZP removal methods (mechanically by splitting with a 28-gauge needle versus chemically by the treatment of acid-Tyrode's solution) and two ICM isolation methods (ZP-free whole blastocyst seeding versus mechanical isolation of ICM) were evaluated for the efficient isolation and culture of putative parthenogenetic bovine ESC. The number of maintained outgrown colonies was counted in each experimental group. As the result, mechanical removal of ZP with a needle and followed by whole ZP-free blastocyst seeding on feeder cells tended to attach more on the feeder layer and resulted in more outgrown colonies with its simple and less time-costing benefits. Currently we are generating ESC lines in HanWoo cattle by using this method for initial outgrowth of the parthenogenetic bovine blastocysts.

• Fisheries and Aquatic Sciences
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• 제19권4호
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• pp.16.1-16.8
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• 2016

To culture germline stem cells in vitro, establishment of the cell lines that can be used as the feeder cells is a prerequisite. In this study, we tried to establish gonad-derived cell lines in Siberian sturgeon (Acipenser baerii). Five 1-year-old A. baerii were used as a donor of gonad tissues, and gonad-dissociated cells were cultured in vitro. Subsequently, determination of growth conditions, long-term culture, characterization, and cryopreservation of the cell lines were also conducted. Five gonad-derived cell lines were stably established and cultured continuously over at least the 73th passage and 402 culture days under the media containing 20 % fetal bovine serum at $28^{\circ}C$. All cell lines consisted of two main cell types based on morphology even if the ratio of the two cell types was different depending on cell lines. Despite long-term culture, all cell lines maintained diploid DNA contents and expression of several genes that are known to express in the A. baerii gonad. After freezing and thawing of the cell lines, post-thaw cell viabilities between 57.6 and 92.9 % depending on cell lines were indentified, suggesting that stable cryopreservation is possible. The results and the cell lines established in this study will contribute to the development of an in vitro system for A. baerii germline stem cell culture.

• 한국가축번식학회지
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• 제25권3호
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• pp.219-225
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• 2001

토끼 배아세포(Embryonic Stem Cell)를 분리하기 위해 토끼 1-cell embryo를 채란하여 in vitro에서 blastocyst까지 배양한 후 mouse embryonic fibroblasts(MEF), rabbit embryonic fibroblasts(REF) 및 STO cell expressing Leukemia Inhibition Factor gene(SNL) feeder cell과 공배양하였다. 외관상 충실한 토끼 배아세포 8 개를 확보하였고 분리된 토끼 ES cell의 모양은 주위에 분화된 세포가 없는 전형적인 colony모양으로 성장하고 액체질소에 동결보존 및 3∼5차례의 계대배양 후에도 이러한 모양은 계속 유지되었다. 충분히 자란 dish를 1 : 2로 계대배양을 한 후 다시 confluent하게 자라는 데에 걸리는 시간(doubling time)은 빠른 경우 84시간으로 나타났다. 분리 된 토끼 ES cell은 gelatin이 coating되지 않은 culture dish에 이식 배양하였을 때 부유상태로 증식하면서 내부에 강이 생기고 외배엽과 내배엽이 형성하는 전형적인 Embryoid body 모양을 나타내어 분리된 ES cell이 미분화상태의 stem cell임이 확인되었다. 본 연구를 통해 토끼에서의 수정란 배양을 통해 토끼 배아세포를 분리하여 특성을 규명하였다 현재까지의 연구성과로는 토끼 수정란의 배양기술을 완벽하게 개발했다는 점과 토끼에서 ES cell을 분리하여 앞으로 유전자 조작의 가능성을 열어 놓은 것이다. 토끼 ES cell system이 완벽히 확립되도록 분리된 ES cell에 대한 미분화상태의 연구 및 미분화상태를 식별할 수 있는 marker등에 대한 연구에 이용될 것이고 복제토끼 및 형질전환토끼의 생산 등을 위한 연구에 이용될 수 있다.

• 전기학회논문지
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• 제64권2호
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• pp.342-348
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• 2015

In this paper, fuel cell power generation system that is being studied in recent railway field was applied to compensate for the voltage drop due to the load as driving electric vehicle. PSIM simulation program is to be used to implement the modeling of the electric railway for AC AT feeder system. For it, It was applied to the product-type single-phase PLL algorithm, step-down converter is controlled as power so as to have the fuelcell generation system. Based on it's result, a reactive power due to the catenary impedance in accordance with the current flowing is compensated as linked with fuelcell generation system which supplied the current to the power supply grid. and then its performance was confirmed that voltage compensation effect obtained at SubStation (SS), SubSectioning Post (SSP), Sectioning Post (SP).

• 식물조직배양학회지
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• 제27권2호
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• pp.125-131
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• 2000

원형질체는 현탁배양된 세포괴로부터 1% cellulase R-10, 0.25% pectolyase Y-23, 그리고 0.5% driselase가 포함된 CPD 효소용액에서 4시간의 진탕 (40 rpm)조건에서 쉽게 분리되었다. 분리된 원형질체는 1 mg/L 2,4-D, 0.5 mg/L kinetin , 200 rng/L spermidine 그리고 68 g/L glucose가 포함된 KM 액체배지에서 배양되었으며, 또한 현탁배양중인 동종의 feeder cell이 있는 CP배지 위에 액체배양배지를 1 mL균일하게 퍼트린 후,$25^{\circ}C$, 암조건으로 4주간 양육배양하였다. 원형질체 유래 SF 캘러스는 0.05 mg/L IAA, 7 mg/L 2lip와 30mg/L sucrose가 첨가된 MS 재분화 배지에서 광조건으로 4주동안 배양되었을 때, 60% 이상의 shoot를 형성하였다. 이후, 식물의생장조절물질이 첨가되지 않은 MS 배지로 옮겨 뿌리를 유도하였으며, 재분화된 식물체는 토양으로 옮겨 배양 8주후에 종자를 형성하였다.

• Asian-Australasian Journal of Animal Sciences
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• 제9권5호
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• pp.583-590
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• 1996

The purpose of this study was to evaluate some factors in the bovine embryonic development from one-cell to blastocyst using modified synthetic oviduct fluid medium (mSOFM), after maturation and in vitro fertilization of the oocytes. The embryonic development to the blastocyst stage was assessed at 7-10 days after in vitro fertilization, and the total cells in the blastocysts were counted by staining nuclei with fluorochrome. Some commercial calf sera (CS) and a superovulated cow serum had different effects on the embryonic development to the blastocyst stage (8.6-21.4%), dependent upon their product lots, although the development might not be affected at least by serum progesterone levels. ${\beta}$-Mercaptoethanol (${\beta}$-ME) supplemented into mSOFM was effective to the embryonic development (27.8%), as well as the co-culture system with cumulus cells (19.5%). In a serum- and feeder cell-free culture using mSOFM containing several growth factors and ${\beta}$-ME instead of CS plus co-cultured cumulus cells, bovine serum albumin (BSA, fraction V), but not polyvinyl alcohol (PVA), was highly effective in embryonic development to the blastocyst stage, almost comparable to CS in the serum-contained culture (CS, BSA and PVA; 27.8, 19.5 and 5.7%, respectively). However, fatty acid free BSA rather reduced the number of developed blastocysts, compared with fraction V BSA (7.3 vs 29.4%). In the serum- and feeder cell-free culture, supplement of glucose to the medium (final 2.0 mM) stimulated the cell proliferation of developing embryos 120 hr after in vitro fertilization. These results indicated that a serum-free medium supplemented with ${\beta}$-ME could successfully support the development of bovine one-cell embryos to the blastocyst stage. Moreover, supplement of glucose and fatty acids to the medium might support preferably the development and cell proliferation of embryos.

• 한국가축번식학회지
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• 제18권4호
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• pp.235-244
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• 1995

생쥐 배반포기 내부세포괴를 체외에서 분리 배양하여 분화가 억제된 내부세포괴 유래 증식세포를 미분화 상태에서 무한히 증식할 수 있는 전능성을 지닌 배아간세포(embryonic stem cell : ES cell)로 확립하고자 본 연구를 실시하였다. BCF1 생쥐 배반포를 10% FCS, 0.1mM nonessential amino acid, 0.1mM sodium pyruvate, 0.1mM 2-mercaptoethanol과 1,000U/ml LIF(세포분확억제인자)가 첨가된 DMEM 기초배양액에 mitomycin-C를 처리한 STO 단층배양세포에서 배양하여 분화가 억제된 내부세포괴 유래의 배아간세포를 분리하였다. 배반포를 STO 단층배양세포에서 4일간 배양하여 내부세포괴세포를 신선한 STO 단층배양세포에서 약 5일 간격으로 반복하여 계대배양을 실시하였다. 5차 계대배양후 뚜렷한 분화 양상없이 배양된 미분화 세포군에 대한 alkaline phosphatase (AP)염색과 체외분화능 검색을 실시한 결과 적색의 미분화 AP 양성반응이 확인되었으며 체외에서 배분화 형성이 유도됨에 따라 배양된 배아간세포주의 다능성 배아간세포 특성을 확인할 수 있었다.