• 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.

• Radiation Oncology Journal
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• v.17 no.2
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• pp.158-165
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• 1999

Purpose : To investigate the percentage of colonies wi1h16or more cells distribution of human skin fibroblast according to in vitro aging, and to evaluate the relationship between percentage of colonies with 10 or more cells and in vivo donor age in human skin fibroblast culture. Material and Method : C1, C2, C3a, and C3b human skin fibroblast samples from three breast cancer patients were used as subjects. The C1, C2, and C3a donor were 44, 54, and 55 years old, respectively. C3a and C3b cells were isolated from the same person. Single cell suspension of skin fibroblasts was prepared with primary explant technique. One hundred cells are plated into 100m1 tissue culture flask and cultured for two weeks. The colony size was defined as colonies with 16 or more cells. The cultured cell was stained with crystal violet, and number of cells in each colony was determined with stereo microscope at $\times$10 magnification. Passage number of C1, C2, C3a and C3b skin fibroblast were 12th, 17th, and 14th, respectively. Results : Percentage of colonies with 16 or more cells of skin fibroblast samples decreased with increasing in vitro passage number. In contrast, cumulative population doublings of skin fibroblast sample increased with increasing in vitro passage number. Percentage of colonies with 16 or more cells also decreased with increasing population doublings in human skin fibroblast culture. There was strong correlation with percentage of colonies with 16 or more cells and population doublings En C3a skin fibroblast sampie. At the same point of population doublings, the percentage of colonies with 16 or more cells of the young C1 donor was higher level than the old C3a donor. Conclusion : The population doublings increased with increasing in vitro passage number but percentage of colonies with 16 or more cells decreased. The results of this study imply that percentage of colonies with 16 or more cell is useful as a indicator of in vitro human skin fibroblast aging and may estimate the in vivo donor age.

• Proceedings of the KSAR Conference
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• 2001.03a
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• pp.22-22
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• 2001

To produce reconstituted rabbit embryos with fetal fibroblasts, the present study was evaluated the efficiencies of the different fusion and 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% $CO_2$ in air. The culture was maintained for 10 passages. In every passage half of cell suspension were kept In frozen. From rabbits treated with FSH in 30% PVP solution and hCG, oocytes were surgically collected from oviducts at 14 h post-hCG injection and stripped off their cumulus cells by re-pipetting in a 300 IU hyaluronidase solution. Oocytes with an extruded first polar body and dense cytoplasm were enucleated by micromanipulation in Ham's F-10 medium＋7.5 g/$m\ell$ cytochalasin B. Euncleation was confirmed under a fluorescence microscope after staining with 5 g/$m\ell$ bisbenzimide for 2 min. Each enucleated oocyte was injected with a fetal fibroblast into a perivitelline space. Reconstructed eggs were compared fusion rates either at 2.0 ㎸/cm or 1.6 ㎸/cm(60 sec, double pulses). After fusion, all eggs were activated with the combination of 5 M ionomycin (5 min) and 10 g/$m\ell$ cycloheximide (CHX, 3h), and cultured in CRlaa medium and transferred into TCM199＋10% FBS on day 3. Although there was not significantly differ in fusion rate between treatments (60%, 2.0 ㎸/cm vs. 79.4%, 1.6 ㎸/cm), none of them in the eggs fused with 2.0 ㎸/cm developed to blastocyst. In comparison of development and chromosome status between different activation treatments (Group 1; 5 M ionomycin/10 g/$m\ell$ CHX, Group 2; 5 M ionomycin/5 g/$m\ell$ CHX ＋ 2 mM DMAP after fusion with 1.6 ㎸/cm), there were not differ in cleavage and development rates (67.3% and 28.9% in Group 1; 67% and 33% in Group 2). All out of 8 embryos evaluated in Group 1 appeared a normal diploid chromosome sets and mean number of cells (Mean SEM) on day 4.5 of culture was 141.5 23.15 (n=8). It can be concluded that the use of cycloheximide has not happened in chromosome abnormalities, and fetal fibroblasts can be used for cloning in rabbit.

• The Korean Journal of Pesticide Science
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• v.12 no.3
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• pp.291-294
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• 2008

The Streptomyces padanus isolate TH04, isolated from mummified peaches, showed strong antifungal activity to Monilinia fructicola. The inhibition activity of the isolate TH04 to mycelial growth and spore germination at 1% concentration of sub-antifungal powder made from culture suspension (CS) was ranged from 79.8% to 81.0% and from 73.9% to 75.8% to M. fructicola four strains, respectively. In the test of antifungal activity in mixed culture of the isolate and M. fructicola, inhibition rate was 7.5%, 86.8% and 94.0% in 0.01, 0.1, and 1% concentration of CS containing bacterial cell of the isolate, respectively. On apples (cultivar; Fuji), the control values of the isolate TH04 crude filtrates (0.1 and 1%) were 85.9% and 100%, respectively. The results suggest that the isolate TH04 indicate development possibility as biocontrol agent of brown rot caused by M. fructicola with the study on delivery method and fermentation condition to produce an antifungal compound.

• Reproductive and Developmental Biology
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• v.30 no.1
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• pp.47-52
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• 2006

This study was conducted to examine whether the parthenogenetic mouse embryonic stem (P-mES) cells can differentiate into functional cardiomyocytes in vitro similar to (mES) cells. p-mES04 and IVF-derived mES03 cells were cultured by suspension culture for 4 days. The formed embryoid bodies (EBs) were treated with 0.75% dimethyl-sulfoxide (DMSO) for further 4 days (4-/4+), and then plated onto gelatin coated culture dish. The appearance of contracting cardiomyocytes from the P-mES04 and mES03 cells was examined for 30 days. The highest cumulative frequency was detected at days 13 (69.83%) and 22 (61.3%), respectively. By immunocytochemistry, beating P-mES04 cells were positively stained with muscle specific anti-sarcomeric a-actinin Ab and cardiac specific anti-cardiac troponin I Ab similar to contracted mES03 cells. When the expression of cardiac muscle-specific genes was analyzed by RT-PCR, beating P-mES04 cells were expressed cardiac specific L-type calcium channel, a1C, cardiac myosin heavy chain a, cardiac muscle heavy polypeptide $7{\beta}$, GATA binding protein 4 and atrial natriuretic factor, but not expressed skeletal muscle specific L-type calcium channel, a1S, which was similar to male adult heart cells and mES03-derived beating cardiomyocytes. The result demonstrates that the P-mES cells can be used as an alternative for the study on the characteristic analysis of in vitro cardiomyocyte differentiation from the ES cells.

• 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 Plant Biotechnology
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• v.32 no.1
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• pp.31-35
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• 2005

To obtain regeneration system of onion, we analyzed the effects of 2,4-D and BA concentration on the embryogenic callus induction from mature embryos. The highest embryogenic callus induction ratio was shown on MS medium (Murashie and Skoog 1962) containing $2.5\;\cal{mg/L}\;or\;5\;\cal{mg/L}$ picloram after mature embryos were placed on medium. When induced callus were cultured on half strength of MS medium containing $1\;\cal{mg/L}$ Kinetin, the highest shoot formation ratio was observed on MS medium containing $1\;{mg/L}$ 2,4-D and $1\;{mg/L}$ BA. Embryogenic callus were cultured in MS liquid medium containing $1\;\ccal{mg/L}$ of 2,4-D and $1\;\cal{mg/L}$ BA. The suspension cultured cell clumps could be mass propagated. Embryogenic callus were friable, but non-embryogenic callus included a lot of moisture, hence the identification between embryogenic and non-embryogenic callus as easily achieved. When embryogenic callus as cultured on half strength of MS medium containing $1\;\cal{mg/L}$ Kinetin, shoots were induced. The whole plantlet was obtained on rooting medium containing $0.5\;\cal{mg/}$ of NAA.

• Research in Plant Disease
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• v.26 no.1
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• pp.8-18
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• 2020

Red pepper, one of the major economic crops in Korea, is being affected by anthracnose disease caused by Colletotrichum acutatum. To control this disease, an antagonistic bacterial strain, Bacillus subtilis YGB36 identified by 16S rDNA sequencing, physiological and biochemical analyses is used as a biological control agent. In vitro screening revealed that the strain YGB36 possess strong antifungal activity against the pathogen Cylindrocarpon destructans. The strain exhibited cellulase, protease, amylase, siderophore production and phosphate solubility. In vitro conidial germination of C. acutatum was most drastically inhibited by YGB36 cell suspensions (10⁶ cfu/ml) or culture filtrate. Development of anthracnose symptoms was reduced on detached immature green pepper fruits by treatment with cell suspensions, and its control value was recorded as 65.7%. The YGB36 bacterial suspension treatment enhanced the germination rate of red pepper seeds and promoted root development and growth under greenhouse conditions. The in vitro screening of fungicide and insecticide sensitivity test against YGB36 revealed that the bacterial growth was not affected by any of the insecticides, and 11 fungicides out of 21 used. Collectively, our results clearly suggest that the strain YGB36 is considered as one of the potential biocontrol agents against anthracnose disease in red pepper.

• Reproductive and Developmental Biology
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• v.36 no.1
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• pp.33-37
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• 2012

Differential capacity of the parthenogenetic embryonic stem cells (PESCs) is still under controversy and the mechanisms of its neural induction are yet poorly understood. Here we demonstrated neural lineage induction of PESCs by addition of insulin-like growth factor-2 (Igf2), which is an important factor for embryo organ development and a paternally expressed imprinting gene. Murine PESCs were aggregated to embryoid bodies (EBs) by suspension culture under the leukemia inhibitory factor-free condition for 4 days. To test the effect of exogenous Igf2, 30 ng/ml of Igf2 was supplemented to EBs induction medium. Then neural induction was carried out with serum-free medium containing insulin, transferrin, selenium, and fibronectin complex (ITSFn) for 12 days. Normal murine embryonic stem cells derived from fertilized embryos (ESCs) were used as the control group. Neural potential of differentiated PESCs and ESCs were analyzed by immunofluorescent labeling and real-time PCR assay (Nestin, neural progenitor marker; Tuj1, neuronal cell marker; GFAP, glial cell marker). The differentiated cells from both ESC and PESC showed heterogeneous population of Nestin, Tuj1, and GFAP positive cells. In terms of the level of gene expression, PESC showed 4 times higher level of GFAP expression than ESCs. After exposure to Igf2, the expression level of GFAP decreased both in derivatives of PESCs and ESCs. Interestingly, the expression level of $Tuj1$ increased only in ESCs, not in PESCs. The results show that IGF2 is a positive effector for suppressing over-expressed glial differentiation during neural induction of PESCs and for promoting neuronal differentiation of ESCs, while exogenous Igf2 could not accelerate the neuronal differentiation of PESCs. Although exogenous Igf2 promotes neuronal differentiation of normal ESCs, expression of endogenous $Igf2$ may be critical for initiating neuronal differentiation of pluripotent stem cells. The findings may contribute to understanding of the relationship between imprinting mechanism and neural differentiation and its application to neural tissue repair in the future.

• Parasites, Hosts and Diseases
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• v.23 no.2
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• pp.293-299
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• 1985

This study is to verify the protective ability against experimental Naegleria meningoencephalitis by immunization with Naegleria fowleri in mice. Naegleria fewleri, strain 0359, and Naegleria gruberi, strain EGB, were used in this study, and cultured in CGVS medium akenically. Inbred BALB/C mice, weighing about 20g, were immunized by three intraperitoneal injection of $1{\times}10^6$ N. fowleri trophozoites at the interval of one week. This N. fowleri trophozoites antigen was fixed with 5% formaldehyde. N. fowleri trophozoites from culture were homogenized with soiicator at $4^{\circ}C$ as monitored by phase contrast microscopy, and their membrane and cell content preparations were made for the immunization of mice. Their inoculation dose in volume was equivalent to the $1{\times}10^6$ trophozoites in each injection for immunization. And N. gruberi trophosoites, whieh was fixed with 5% formaldehyde, were also used for immunisation. Mice were inoculated intranasally with $5{\times}10^4$ N. fowleri trophozoites in a 511 suspension under anesthesia by as intraperitoneal injection of about 1 mg secobarbiturate. Nervousness, rotation or sluggish behaviour were observed in the mice which were infected with N. fewleri. Necrotic lesion was demonstrated in the anterior portion of brain, especially in the olfactory lobe. The inflammatory cell infiltration with numerous H. fowleri trophozoites was noticed. This pathological changes were more extensive in the control than in the experimental groups. Mice were dead due to experimental primary amoebic meningoencephalitis that developed between 8 days and 23 days after inoculation. Mortality rate of the mice was low in the immunized experimental group. Mean survival time, which is the survival duration of mice from the infection to death, was prolonged significantly in the immunized mice except in the mice immunized with JV, fowleri membrane. Even in the mice immunized with N. gruberi, survival time was delayed. In summary, the effectiveness of immunization is demonstrated in terms of protective immunity against Naegleria meningoencephalitis in mice.