• Journal of Animal Reproduction and Biotechnology
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• v.39 no.1
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• pp.19-30
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• 2024

Background: Although an understanding of the proliferation and differentiation of fish female germline stem cells (GSCs) is very important, an appropriate threedimensional (3D) research model to study them is not well established. As a part of the development of stable 3D culture system for fish female GSCs, we conducted this study to establish a 3D aggregate culture system of ovarian cells in marine medaka, Oryzias dancena. Methods: Ovarian cells were separated by Percoll density gradient centrifugation and two different cell populations were cultured in suspension to form ovarian cell aggregates to find suitable cell populations for its formation. Ovarian cell aggregates formed from different cell populations were evaluated by histology and gene expression analyses. To evaluate the media supplements, ovarian cell aggregate culture was performed under different media conditions, and the morphology, viability, size, gene expression, histology, and E2 secretion of ovarian cell aggregates were analyzed. Results: Ovarian cell aggregates were able to be formed well under specific culture conditions that used ultra-low attachment 96 well plate, complete mESM2, and the cell populations from top to 50% layers after separation of ovarian cells. Moreover, they were able to maintain minimal ovarian function such as germ cell maintenance and E2 synthesis for a short period. Conclusions: We established basic conditions for the culture of O. dancena ovarian cell aggregates. Additional efforts will be required to further optimize the culture conditions so that the ovarian cell aggregates can retain the improved ovarian functions for a longer period of time.

• Korean Journal of Animal Reproduction
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• v.18 no.4
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• pp.299-307
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• 1995

The goal of cell stem cell technology is to produce a viable and genetically normal animal. To achieve this goal various laboratories have followed 2 different pathways beginning with either the culture of 1) single or pooled ICMs grown with or without a feeder layer or 2) single or pooled 16-20 cell stage embryos grown with a feeder layer. Also, thus far embryonic cell cultures or lines have been established by several methods including loose suspension culture for short-term cultures and more commonly murine or bovine fibroblast feeder layers for long-term culture. Pluripotent lines have been derived from 16-cell through blastocyst inner cell mass stages. The efficiency of establishing cell lines and cell proliferation apper to be affected by the number of cells or embryos starting the line. Most attempts to produce offspring from long term STO cell feeder layer cultured ICM or morulae derived ES cells have resulted in pregnancy failure in the first trimester when ES cells were used in cuclear transfer or have failed to retain ES cells in the progeny produced by chimerization. The exception is 1 chimeric fetus from use of morula ES cells in the chimerization with early embryonic cells. There is much to be learned yet about ES cell culture requirements for maintenance of totipotency. If bovine ES cell lines loose imprinting pattern and totipotency with long-term culture and passage as suggested for mouse ES cells, we may be limited to the use of short-term cultures for multiplication of embryos and efficient production of transgenic animals. No bovine ES cell system has yet met all of the criteria indicated for a totipotent ES cell line.

• The Journal of the Korean Society for Microbiology
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• v.22 no.1
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• pp.23-33
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• 1987

To detect lepotospiral strains which produce hemolysin and determine the optimal condition for assaying hemolysin, we screened reference strains and observed some property of hemolysin. Hemolysin activity was assayed with cell free culture liquids and erythrocyte suspension. The production of hemolysin by local strains isolated in Korea was assayed and compared with that of reference strains. The hemolysin was produced by 18 strains among 38 reference strains and 3 local strains isolated in Korea. The production of hemolysin began with growth of Leptospira cultured in EMJH medium and reached maximum at stationary phase. The optimum temperature for hemolytic activity was $37^{\circ}C$. At lower temperature the activity of hemolysin was decreased progressively. The hemolytic activity was completely inactivated after :30 minutes' exposure at $56^{\circ}C$. Hemolysis pattern was "hot-cold type" which showed increased hemolysis after cold incubation. The hemolysin was most active on sheep erythrocyte and less active on ox, goat, human and guinea pig erythrocyte with the decreasing order.

• Proceedings of the KSAR Conference
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• 2001.10a
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• pp.60-61
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• 2001

To produce reconstituted rabbit embryos with fetal fibroblasts, the present study was evaluated the efficiencies of the activation conditions as assessments of subsequent development and chromosome in the embryos. New Zealand White rabbits were used throughout the study. Fetal fibroblasts collected from 22-d of fetuses were cultured in DMEM＋10% FBS in 5% CO₂ in air. The culture was maintained for 10 passages. In every passage half of cell suspension were kept in frozen. (omitted)

• BMB Reports
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• v.30 no.6
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• pp.379-384
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• 1997

The effects of several ions on the specific activity and isozyme patterns of cellular and secretory isoperoxidases were studied in suspension-cultured cells of rice (Oryza sativa L.). Peroxidase release into the culture medium occurred in the absence of added calcium. The addition of calcium ion greatly stimulated the secretion of cationic isoperoxidases such as C2 and C3 into the medium: a maximum 11 fold increase of secretions occurred in the presence of 5 mM $CaCl_2$, and the secretion was accomplished within 1 hour after the addition of $CaCl_2$. About a 10 fold increase of the peroxidase secretion into the medium did occur with 0. 5% NaCl, whereas cellular isoperoxidase levels were reduced notably. About a 6 fold increase of the specific activity of cellular isoperoxidase was found in 5 mM $NiCl_2$-treated cell, while $NiCl_2$ had no effect on the secretion of peroxidase into the medium. Various concentrations of KCl did not change peroxidase secretion, but 5 mM $ZnCl_2$ reduced peroxidase secretion greatly. The major secretory isoperoxidases stimulated by $CaCl_2$, NaCl and cellulase were composed of cationic isoperoxidases C2 and C3, which were found to be localized in the cell wall of rice by examination of the enzyme in the protoplast. Furthermore, the secretion rates of secretory isoperoxidases were increased rapidly when cellulase was treated in the absence of the osmotic stabilizer of 0.4 M mannitol. These results suggest that the stimulations of secretory isoperoxidase levels seem to be due to the stimulation of secretion into the culture medium of rice.

• Korean Journal of Animal Reproduction
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• v.14 no.1
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• pp.1-8
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• 1990

These experiments were carried out to investigate the effects of cumulus cells for in vitro fertilization and development of bovine follicular oocytes matured in vitro. The bovine ovaries were obtained at a slaughter house and the follicular oocytes surrounded by cumulus cells were collected by puncturing follicles with 2~6 mm of diameter. Bovine oocytes were matured in vitro for 24~26 hours in a CO2 incubator with 5% CO2 in air at 39$^{\circ}C$. The medium used for maturation was TCM-199 supplemented with hormones, pyruvate, FCS and antibiotics. Epididymal spermatozoa were capacitated by in vitro culture for 2~3 hours in BO solution containing BSA(5mg/ml) and caffeine(2.5mM). Insemination was made by introducing about 10~15 matured oocytes into the suspension of capacitated spermatozoa. Six hour after insemination the eggs were transferred to TCM-199 supplemented with FCS(10%) and HEPES(25mM), cultured for 7~8 days with 10~15 eggs/well in 4-well multidishes(Nunc Co.) forming cumulus cell monolayer. The results obtained in these experiments were summarized as follows ; 1. The majority of the follicular oocytes with compacted cumulus cells existed in GV stage while those with dispersed or denuded cumulus cells existed GVBD and M II stage. 2. After 24~26 hours maturation, the maturation rates of the follicular oocytes cultured in TCM-199 containing hormones were slightly higher than those of oocytes cultured in medium without hormones, and the frequency of cumulus compacted or denuded oocytes reaching M II stage cultured in medium containing hormones was 75.7% or 51.7%, respectively(P<0.05). 3. After 20 hours in vitro insemination, percentages of ova fertilized were 61.4% or 51.4%, respectively, for cumulus oophorus intacted or removed, and increased frequency of ova with both male and female pronuclei was found when cumuli were present(P<0.05). 4. The rates of embryos developed to 2-, 4-, 8-, 16-cell and morula or blastocyst stage after cocultured with cumulus cells were 65.0%, 45.3%, 34.7%, 28.0% and 22.7%, respectively. The results for momla or blastocyst stage were significantly higher than those of the embryos cultured in the basic medium(P<0.05).

• Journal of Periodontal and Implant Science
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• v.26 no.1
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• pp.1-26
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• 1996

This study was performed to estimate the effects of cultured bone cell inoculated on porous type hydroxyaptite for the regeneration of the artificial alveolar bone defect. In this experiment 3 beagle dogs were used, and each of them were divided into right and left mandible. Every surgical intervention were performed under the general anesthesia by using with intravenous injection of Pentobarbital sodium(30mg/Kg). To reduce the gingival bleeding during surgery, operative site was injected with Lidocaine hydrochloride(l:80,000 Epinephrine) as local anesthesia. After surgery experimental animal were feeded with soft dietl Mighty dog, Frisies Co., U.S.A.) for 1 weeks to avoid irritaion to soft tissue by food. 2 months before surgery both side of mandibular 1st premolar were extracted and bone chips from mandibular body were obtained from all animals. Bone cells were cultured from bone chips obtained from mandible with Dulbecco's Modified Essential Medium contained with 10% Fetal Bovine Serum under the conventional conditions. Porous type hydroxyapatite were immerse into the high concentrated cell suspension solution, and put 4 hours for attachin the cells on the surface of hydroxyapatite. Graft material were inserted on the artificial bone defect after 3 days of culture. Before insertion of cellinoculated graft material, scanning electronic microscopic observation were performed to confirm the attachment and spreading of cell on the hydroxyapatite surface. 3 artificial bone defects were made with bone trephine drill on the both side of mandible of the experimental animal. First defect was designed without insertion of graft material as negative control, second was filled with porous replamineform hydroxyapatite inoculated with cultured bone marrow cells as expermiental site, and third was filled with graft materials only as positive control. The size of every artificial bone defect was 3mm in diameter and 3mm in depth. After the every surgical intervention of animals, oral hygiene program were performed with 1.0% chlorhexidine digluconate. All of the animals were sacrificed at 2, 4, 6 weeks after surgery. For obtaining histological section, tissus were fixed in 10% Buffered formalin and decalcified with Planko - Rycho Solution for 72hr. Tissue embeding was performed in paraffin and cut parallel to the surface of mandibular body. Section in 8um thickness of tissue was done and stained with Hematoxylin - Eosin. All the specimens were observed under the light microscopy. The following results were obtained : 1. In the case of control site which has no graft material, less inflammatory cell infiltration and rapid new bone forming tendency were revealed compared with experimental groups. But bone surface were observed depression pattern on defect area because of soft tissue invasion into the artificial bone defect during the experimental period. 2. In the porous hydroxyapatite only group, inflammatory cell infiltration was prominet and dense connective tissue were encapsulated around grafted materials. osteoblastic activity in the early stage after surgery was low to compared with grafted with bone cells. 3. In the case of porous hydroxyapatite inoculated with bone cell, less inflammatory cell infiltration and rapid new bone formation activity was revealed than hydroxyapatite only group. Active new bone formation were observed in the early stage of control group. 4. The origin of new bone forming was revealed not from the center of defected area but from the surface of preexisting bony wall on every specimen. 5. In this experiment, osteoclastic cell was not found around grafted materials, and fibrovascular invasion into regions with no noticeable foreign body reaction. Conclusively, the cultured bone cell inoculated onto the porous hydroxyapatite may have an important role of regeneration of artificial bone defects of alveolar bone.

• Applied Biological Chemistry
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• v.48 no.4
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• pp.425-430
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• 2005

[${\beta}-sitosterol$] is a plant sterol that reduces cholesterol levels and inhibits the growth of human prostate and colon cancer cells. Optimal conditions for ${\beta}-sitosterol$ production were examined from cell suspension cultures of Chrysanthemum coronarium L. The callus induction was optimal in MS medium containing 1 mg/l NAA and 1 mg/l BAP. Cell suspension culture was also established from the callus. Optimal ${\beta}-sitosterol$ production was obtained when the cells were cultured at an initial density of 2 mg DCW/l in MS medium containing 1 X sucrose (30 mg/l), 1 X nitrogen (1900 mg/l $KNO_3$, 1650 mg/l $NH_4NO_3$), and 1 X phosphate source (170 mg/l). In cell suspension cultures of C. coronarium L. using shake flasks, the peak content of ${\beta}-sitosterol$ was $150{\mu}g/g$ DCW. In cell suspension cultures of C. coronarium L. using an air-lift bioreactor, the maximum ${\beta}-sitosterol$ content of $143.8{\mu}g/g$ DCW was obtained at an air-flow rate of 100 cc/min.

• Molecules and Cells
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• v.37 no.6
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• pp.497-502
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• 2014

Microfluidics can provide unique experimental tools to visualize the development of neural structures within a microscale device, which is followed by guidance of neurite growth in the axonal isolation compartment. We utilized microfluidics technology to monitor the differentiation and migration of neural cells derived from human embryonic stem cells (hESCs). We co-cultured hESCs with PA6 stromal cells, and isolated neural rosette-like structures, which subsequently formed neurospheres in suspension culture. Tuj1-positive neural cells, but not nestin-positive neural precursor cells (NPCs), were able to enter the microfluidics grooves (microchannels), suggesting that neural cell-migratory capacity was dependent upon neuronal differentiation stage. We also showed that bundles of axons formed and extended into the microchannels. Taken together, these results demonstrated that microfluidics technology can provide useful tools to study neurite outgrowth and axon guidance of neural cells, which are derived from human embryonic stem cells.

• Korean Journal of Animal Reproduction
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• v.24 no.4
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• pp.385-394
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• 2000

When porcine primordial germ cells (PGCs) isolated from the genital ridge and placed in culture to establish EG cells, a large proportion of PGCs are lost during the early period of culture. To characterize the in vitro death of porcine PGCs, PGCs were cultured in suspension, and apoptosis analyzed using a fluorescent activated cell sorter-based DNA fragmentation assay. The results from flow cytometric analysis showed an increase in apoptosis in cultured cells. However, the cells isolated from the genital ridges are a mixture of PGCs and somatic cells. To detect apoptotic signals specific from porcine PGCs, quantitative TUNEL assay was performed at different time of culture (0 ∼ 24 h). The proportion of apoptotic porcine PGCs determined by double staining with alkaline phosphatase activity and in situ TUNEL assay increased as the time of culture progressed and continued at least 24 h. These results demonstrate that one of the causes of loss of porcine PGCs in vitro is apoptosis.