• 생약학회지
• /
• 제28권4호
• /
• pp.286-292
• /
• 1997

We studied the screening condition for immune regulatory responsor. We focused on the T-lymphocytes leer this purpose. Mouse fetal thymic organ culture (FTOC) system and flow cytometric analysis were mainly used in this experiment. Even if FTOC is carried out in vivo condition, the pattern of thymic development in the condition of FTOC is similar to that of in vivo condition. In this regard, FTOC system might be very powerful tool to screen the immune regulator, especially concerning on T cells. To establish the optimum condition of FTOC to screen the Immune regulator, we focused on the optimum amount of dose and culture period. The cell number and surface antigens on T cells were also analysed by using hemacytometer and flow cytometer. To monitor the differentiation event, anti-CD3, anti-CD4 and anti-CD8 antibodies were used. Alkoxyglycerol and Phellodendri Cortex were used fur positive and negative control, respectively. Astragalus membranceus was used as test sample. From our analysis, we reached to conclusions that the best dose of extract is $50\;{\mu}g/ml$ of culture medium, the best culture period is for 9 days, and ethanol used as solvent has no toxicity to FTOC.

• 한국수정란이식학회지
• /
• 제28권3호
• /
• pp.273-280
• /
• 2013

The aim of this study was to establish a three dimensional (3D) culture system of endometrial cells and to examine the plasminogen activators (PAs) activity in porcine uterine. The 3D culture system in porcine endometrial cells was composed to mixture 3D gel, stromal cells and epithelial cells. The 3D culture system was used to identify normal structure search as uterine tissue and PAs expression in this study. In results, porcine endometrium epithelial cells forming a top monolayer and endometrium stromal cells developed as fibroblast-like within 3D matrix scaffold. Expression of urokinase-type PA (uPA) and tissue-type PA (tPA) were observed during the 3D culture using immunofluorescence. PA activity in 3D-cultured endometrial cells was no significant difference between the tissue type, but 2D culture system were significantly lower than in 3D-cultured endometrial cells (P<0.05). Therefore, basic system and functional aspect of 3D culture could be established with similar system of endometrium tissue. We suggest that this study was assumed applicable as baseline data to investigate mechanism between porcine uterus cells in vitro.

• Journal of Microbiology and Biotechnology
• /
• 제19권9호
• /
• pp.888-897
• /
• 2009

Lipase diversity in glacier soil was assessed by culture-independent metagenomic DNA fragment screening and confirmed by cell culture experiments. A set of degenerate PCR primers specific for lipases of the hormone-sensitive lipase family was designed based on conserved motifs and used to directly PCR amplify metagenomic DNA from glacier soil. These products were used to construct a lipase fragment clone library. Among the 300 clones sequenced for the analysis, 201 clones encoding partiallipases shared 51-82% identity to known lipases in GenBank. Based on a phylogenetic analysis, five divergent clusters were established, one of which may represent a previously unidentified lipase subfamily. In the culture study, 11 lipase-producing bacteria were selectively isolated and characterized by 16S rDNA sequences. Using the above-mentioned degenerate primers, seven lipase gene fragments were cloned, but not all of them could be accounted for by the clones in the library. Two full-length lipase genes obtained by TAIL-PCR were expressed in Pichia pastoris and characterized. Both were authentic lipases with optimum temperatures of ${\le}40^{\circ}C$. Our study indicates the abundant lipase diversity in glacier soil as well as the feasibility of sequence-based screening in discovering new lipase genes from complex environmental samples.

• Journal of Periodontal and Implant Science
• /
• 제35권3호
• /
• pp.747-760
• /
• 2005

Periodontal therapy has dealt primarily with attempts at arresting progression of disease. however, more recent techniques have focused on regenerating the periodontal ligament having the capacity to regenerate the periodontium. Recombinant human bone morphogenetic protein-7(rhBMP-7) can differentiate the osteoprogenitor cells and induce bone formation. The purpose of this study was to evaluate the effect of BMP-7 on rat periodontal ligament cells differentiation, in vitro. In the control group, cells was cultured with DMEM media. In the experimental groups, cells were cultured with rhBMP-7 in concentration of 10, 25, 50 and 100 ng/ml. Each group was characterized by examining alkaline phosphatase activity at 3 and 5 days of culture and the ability to produce mineralized nodules of rat calvarial cells at 14 days of culture. Synthesis of type I collagen(COL-I), osteocalcin(OCN), and bone sialoprotein(BSP) was evaluated by RT-PCR at 7 days of culture. Activation of Smad proteins and p38 MAP kinase was determined by western blot analysis of the cell lysates. Alkaline phosphatase activity was significantly increased in the concentration of BMP-7 50 ng/ml and 100 ng/ml compared to the control(p<0.05). The mineralized bone nodule formation was greater with addition of 50 ng/ml and 100 ng/ml BMP-7 than the control(p<0.01). In 7 days' culture, the expressions of COL-I, BSP, and OCN was increased by BMP-7 in concentration of 10 $ng/ml{\sim}100$ ng/ml. In western blot analysis, BMP-7 treated culture cells expressed Smad 1,5,8 in dose-dependent manner, whereas BMP-7 did not activate phosphorylated form of p38 MAP kinase. These result suggested that BMP-7 stimulate rat periodontal ligament cells to differentiate toward osteoblast phenotype and increase bone matrix production by activation of BMP-Smad pathway.

• ALGAE
• /
• 제38권4호
• /
• pp.283-294
• /
• 2023

Previous research indicated that free-living sporangial filament keep hollow morph under high-culture density and form bipartite cells under low-culture density, while the following conchospore release was inhibited by high light. Here, we further explored the molecular bases of these affects caused by light and culture density using a transcriptome analysis. Many differentially expressed genes (DEGs) related to carbon dioxide concentration and fixation, photosynthesis, chlorophyll synthesis and nitrogen absorption were upregulated under high-light conditions compared with low-light conditions, indicating the molecular basis of rapid vegetative growth under the former. The stress response- and ion transport-related DEGs, as well as the gene encoding the vacuole formation-brefeldin A-inhibited guanine nucleotide exchange protein (BIG, py05721), were highly expressed under high-density conditions, indicating the molecular basis of the hollow morph of free-living sporangial filaments under high-culture density conditions. Additionally, the brefeldin A treatment indicated that the hollow morph was directly influenced by vacuole formation-related vesicle traffic. Others DEGs related to cell wall components, zinc-finger proteins, ASPO1527, cell cycle and cytoskeleton were highly expressed in the low density with low-light group, which might be related to the formation and release of conchospores. These results provide a deeper understanding of sporangial filaments in Neopyropia yezoensis and related species.

• Asian-Australasian Journal of Animal Sciences
• /
• 제23권9호
• /
• pp.1152-1158
• /
• 2010

In differentiating human embryonic stem (d-hES) cells there are a number of types of cells which may secrete various nutrients and helpful materials for pre-implantation embryonic development. This study examined whether the d-hES could function as a feeder cell in vitro to support mouse embryonic development. By RT-PCR analysis, the d-hES cells revealed high expression of three germ-layered differentiation markers while having markedly reduced expression of stem cell markers. Also, in d-hES cells, LIF expression in embryo implantation-related material was confirmed at a similar level to undifferentiated ES cells. When mouse 2PN embryos were cultured in control M16 medium, co-culture control CR1aa medium or co-cultured with d-hES cells, their blastocyst development rate at embryonic day 4 (83.9%) were significantly better in the d-hES cell group than in the CR1aa group (66.0%), while not better than in the M16 group (90.7%)(p<0.05). However, at embryonic days 5 and 6, embryo hatching and hatched-out rates of the dhES cell group (53.6 and 48.2%, respectively) were superior to those of the M16 group (40.7 and 40.7%, respectively). At embryonic day 4, blastocysts of the d-hES cell group were transferred into pseudo-pregnant recipients, and pregnancy rate (75.0%) was very high compared to the other groups (M16, 57.1%; CR1aa, 37.5%). In addition, embryo implantation (55.9%) and live fetus rate (38.2%) of the d-hES cell group were also better than those of the other groups (M16, 36.7 and 18.3%, respectively; CR1aa, 23.2 and 8.7%, respectively). These results demonstrated that d-hES cells can be used as a feeder cell for enhancing in vitro and in vivo developmental potential of mouse pre-implantation embryos.

• KSBB Journal
• /
• 제14권2호
• /
• pp.188-191
• /
• 1999

본 실험에서는 하이브리도마세포 배양 중 아미노산 량의 변화를 조사하여 세포성장, 세포대사 및 항체생산성에 미치는 아미노산의 영향을 조사하였다. 실험결과에 의하면 vR8 하이브리도 마세포 배양 중 세포성장이 급속히 이루어지는 시점과 최대 세포농도에 도달한 시점에서의 glutamine, arginine, leucine 등 다수 아미노산의 고갈이 관찰되었고, 이후 세포활성이 급속히 저하되었다. 이때 glucose 농도, lactate, armmonia 등의 대사산물 농도 및 pH, DO, 교반속도 등은 적절하게 유지되었기 때문에 이들 요소에 의한 세포성장의 저해는 없었다. 따라서 이들 아미노산의 고갈이 세포활성의 저하에 커다란 영향을 주었다고 생각된다. 한편, 고갈된 아미노산을 첨가한 배지(GC-HY-S2)로 배양을 해본 결과 세포농도를 $2.91\times10^7$cell/mL까지 증가시킬 수 있었다. 즉, 변형배지에서는 세포활성의 저하를 가져오는 세포성장구간을 지나 계속적인 세포성장을 보여 27배 정도 더 높은 최대 세포농도를 보였다. 세포농도의 증가로 specific productivity, volumetric productivity도 각각 1.75배, 56배 증가하였다. 최대 세포농도에서의 아미노산 량을 조사한 결과를 보면 충분한 양의 아미노산이 공급되었음을 알 수 있다. 고농도 하이브리도마세포 배양에서의 일부 아미노산의 고갈은 일반적으로 관찰되는 현상이다. 그러므로 세포를 고농도로 배양하기 위해서는 아미노산 분석을 통해 고갈된 아미노산을 첨가해 주어야 하는데, 아미노산의 요구는 세포주마다 다르므로 배지 최적화과정에서 아미노산 분석에 의한 배지의 개선이 필요하다고 생각된다.

• 한국정보통신학회논문지
• /
• 제15권12호
• /
• pp.2697-2704
• /
• 2011

조직 공학에서는 하이드로 젤 내 3차원 세포 배양 과정에서 세포와 세포간의 공생관계를 관찰하게 되는데, 시간이 경과함에 따라 세포들의 증가나 상호작용, 분화, 사멸되는 과정들을 고배율 공초점 현미경을 이용하여 단층 촬영하고, 촬영된 이미지들로부터 세포의 공생 메커니즘이나 변화 과정을 관찰 및 분석하기 위한 연구가 진행되고 있다. 본 연구에서는 하이드로 젤 내 3차원 세포 배양 과정에서 단층 촬영된 세포 이미지로부터 클러스터 경계선 정보를 추출하기 위한 수준별 잡음 제거 방법과 클러스터 경계선 추출 방법을 제안하였다. 실험을 통해 제안된 방법은 하이드로젤로 인한 좁쌀 모양의 잡음에 뛰어난 잡음 제거 성능을 보여 줄 뿐만 아니라 복잡한 클러스터들에 대해서 도 정확한 클러스터 경계선을 추출함을 보여 주었다.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
• /
• 제30권1호
• /
• pp.1-16
• /
• 2004

The use of artificial nerve conduit containing viable Schwann cells is one of the most promising strategies to repair the peripheral nerve injury. To fabricate an effective nerve conduit whose microstructure and internal environment are more favorable in the nerve regeneration than existing ones, a new three-dimensional Schwann cell culture technique using $Matrigel^{(R)}$. and dorsal root ganglion (DRG) was developed. Nerve conduit of three-dimensionally arranged Schwann cells was fabricated using direct seeding of freshly harvested DRG into a $Matrigel^{(R)}$ filled silicone tube (I.D. 1.98 mm, 14 mm length) and in vitro rafting culture for 2 weeks. The nerve regeneration efficacy of three-dimensionally cultured Schwann cell conduit (3D conduit group, n=6) was assessed using SD rat sciatic nerve defect of 10 mm, and compared with that of silicone conduit filled with $Matrigel^{(R)}$ and Schwann cells prepared from the conventional plain culture method (2D conduit group, n=6). After 12 weeks, sciatic function was evaluated with sciatic function index (SFI) and gait analysis, and histomorphology of nerve conduit and the innervated tissues of sciatic nerve were examined using image analyzer and electromicroscopic methods. The SFI and ankle stance angle (ASA) in the functional evaluation were $-60.1{\pm}13.9$, $37.9^{\circ}{\pm}5.4^{\circ}$ in 3D conduit group (n=5) and $-87.0{\pm}12.9$, $32.2^{\circ}{\pm}4.8^{\circ}$ in 2D conduit group (n=4), respectively. And the myelinated axon was $44.91%{\pm}0.13%$ in 3D conduit group and $13.05%{\pm}1.95%$ in 2D conduit group to the sham group. In the TEM study, 3D conduit group showed more abundant myelinated nerve fibers with well organized and thickened extracellular collagen than 2D conduit group, and gastrocnemius muscle and biceps femoris tendon in 3D conduit group were less atrophied and showed decreased fibrosis with less fatty infiltration than 2D conduit group. In conclusion, new three-dimensional Schwann cell culture technique was established, and nerve conduit fabricated using this technique showed much improved nerve regeneration capacity than the silicone tube filled with $Matrigel^{(R)}$ and Schwann cells prepared from the conventional plain culture method.

• Environmental Analysis Health and Toxicology
• /
• 제18권4호
• /
• pp.295-303
• /
• 2003

Physiological cellular activities responses to cadmium (Cd) exposure in green algae with several reductases activities and viability of the cell were examined. The cell division of green algae, Selenastrum capricornutum treated with 5ppm was significantly decreased than that of normal algae. The mean cell number of normal algal culture was as twice much as than that of algae at 6 days after Cd treatment. The cellular viability of algae was analysed by flow-cytometry with fluorescent dye after esterase reaction on cell membrane. The 85.35% of cellular viability of normal culture was decreased to 34.35% when algae was treated with 5 ppm of Cd at 6 days after treatment. It was considered that those method of flow-cytometry is useful tool for toxicity test on micro-organisms in the respect of identifying cellular viability. Also, the activities of both glutathione peroxidase (GPX) and ascorbate peroxidase (APX), which are indirectly react against oxidative stress through reduction of glutathione by Cd were significantly increased with 25%. It is considered that both GPX and APX are involved in the metabolic pathway of Cd -detoxification with similar portion in Selenasturm capricornutum.