Background: In healthy dentin conditions, odontoblasts have an important role such as protection from invasion of pathogens. In mammalian teeth, progenitors such as mesenchymal stem cells (MSCs) can migrate and differentiate into odontoblast-like cells, leading to the formation of reparative dentin. For differentiation using stem cells, it is crucial to provide conditions similar to the complex and intricate in vivo environment. The purpose of this study was to evaluate the potential of differentiation into odonto/ osteoblasts, and compare co-culture with/without epithelial cells. Methods: MSCs and epithelial cells were successfully isolated from dental tissues. We investigated the influences of epithelial cells on the differentiation process of dental pulp stem cells into odonto/osteoblasts using co-culture systems. The differentiation potential with/without epithelial cells was analyzed for the expression of specific markers and calcium contents. Results: Differentiated odonto/osteoblast derived from dental pulp tissue-derived mesenchymal stem cells with/without epithelial cells were evaluated by qRT-PCR, immunostaining, calcium content, and ALP staining. The expression of odonto/ osteoblast-specific markers, calcium content, and ALP staining intensity were significantly increased in differentiated cells. Moreover, the odonto/osteogenic differentiation capacity with epithelial cells co-culture was significantly higher than without epithelial cells co-culture. Conclusions: These results suggest that odonto/osteogenic differentiation co-cultured with epithelial cells has a more efficient application.

인터넷 시대를 맞아 온라인 콘텐츠 플랫폼은 텔레비전 미디어 시대의 송년 매체 의식을 변화시켰고, 기수 매체의 변화 중의 의식 활동을 분석 하여 새로운 환경을 제공하였다.　따라서 본 연구에서는 온라인 콘텐츠 플랫폼에서 개최하는 송년회를 분석 대상으로 선정하여 온라인 커뮤니티 의 의식 활동을 연구하였다. 이론적인 측면으로 본 연구는 매체 의식의 이론적 관점에서 참여형 문화 이론과 결합하여 커뮤니티의 매체 의식 실 천을 분석하고, 참여형 매체 의식을 사용하여 온라인 콘텐츠 플랫폼에서 커뮤니티의 역할에 응한다. 본 연구는 콘텐츠의 협력 공동창작 및 커뮤 니티 사용자 참여 등 두 가지 관점에서 온라인 커뮤니티의 송년 행사와 다른 미디어 플랫폼이 창출하고 주도하는 송년 행사의 차이점을 분석한 다. 본 연구의 핵심 목적은 온라인 콘텐츠 플랫폼의 송년 활동의 혁신을 분석하고, 온라인 커뮤니티가 매체 의식 활동을 만들고 기획하는 방법을 더 잘 이해하고, 대중문화와 커뮤니티 사용자가 콘텐츠 공동창출을 통해 커뮤니티의 단결을 만들고 커뮤니티의 응집 효과를 강화하며 상호 작용 에 참여하여 의식 상호 작용을 촉진할 수 있는 아이디어를 제공하는 것 이다.　본 연구에서는 콘텐츠 창작 과정에서 온라인 콘텐츠 플랫폼에서 커뮤니티 네트워크의 주도적 위치를 지적했다. 따라서 콘텐츠 서사 단계 에서는 시청자에게 주의를 기울이고 매체 전반에 걸쳐 참여감을 부여하 고 플랫폼은 미디어 의식의 장이자 중개자가 된다.

In pig, more than half of the recovered cumulus cell-oocyte complexes (COCs) have one or two layers of cumulus cells and are considered morphologically poor. If we could take full advantage of these poor quality COCs, we could potentially improve the efficiency of in vitro embryo production. During in vitro maturation, although some maturation factors are transmitted bidirectionally between the oocyte and cumulus cells of the same COC, transmission also occurs between different COCs. We hypothesized that morphologically poor COCs fail to undergo complete oocyte maturation due to their insufficient secretion of maturation factors. Here, we investigated whether co-culture with morphologically good COCs (having three or more layers of cumulus cells) could improve the maturation and utilization rates of morphologically poor COCs. Our results revealed that the oocyte maturation rate, glutathione level, embryo development capacity, blastocyst quality, and cumulus cell gene expression levels of BCL-2 and PCNA were similar in the co-culture and good quality-groups, and that these levels were all significantly higher than those in the poor quality-group. Our results strongly suggest that the co-culture strategy greatly improved the utilization rate of morphologically poor COCs without reducing their capacity for maturation and subsequent development.

Carbon source, an essential nutrient for plant growth, mainly includes exogenous sugar and CO2 of the environment in vitro. Therefore, the exogenous sugar and CO2 of the environment make the important roles in tissue culture. The aim of this study is to investigate the effects of different sugar concentrations (0, 10, 15 and 30 g·L-1) on the growth of colored Zantedeschia in vitro under certain CO2 concentration and explore the optimal sugar concentration. The plantlets in vitro of colored Zantedeschia had the largest root number, root weight, and root vigor under 0 g·L-1 (sugar-free culture) treatment. And they had the largest plant height, leaf length and leaf chlorophyll content, but p oor r oot v igor under 3 0 g·L-1 sugar. This study indicated that the optimal condition for proliferation and seedling culture of colored Zantedeschia plantlets in vitro was MS medium with 30 g·L-1 sugar, and the suitable medium for rooting culture and transplanting of colored Zantedeschia was MS medium with sugar-free culture under CO2 enrichment condition.

In order to achieve successful in vitro production of embryo, it is necessary to establish intrauterine environment during in vitro culture. Thus, this study was investigated to establish embryo culture system using co-incubated collagen matrix gel (CM) with endometrial epithelial cells (EC). Endometrial epithelial cells were isolated from porcine endometrium at follicular phase, the cells seeded in insert dish for co-incubation with CM-coated culture dish. Then, culture media treated with/without 2.0 IU/ml hCG or 10 ng/ml IL-1β. After incubation for 24 h, the co-incubated insert dishes were removed from CM-coated culture dish before embryo culture. Embryos at 48 h after in vitro fertilization (IVF) were cultured on the dish for 120 h with porcine zygote medium. We determined PTGS-2 expression in the ECs, VEGF protein in co-incubated CM with EC and observed cleavage rate and blastocyst development of embryos at 168 h after IVF. In result, expression of PTGS-2 was higher at co-incubated EC with hCG and IL-1β groups than EC without hCG and IL-1β. The VEGF protein was detected at co-incubated CM with EC, EC treated with hCG and IL-1β groups higher than CM group. Also, cleavage rate was no significantly difference among all group, however, blastocyst development was significantly higher in co-incubated CM with EC treated with hCG group than un-treated groups (p<0.05). Therefore, we suggest that novel embryo culture system using co-incubated collagen matrix gel with endometrial epithelial cells treated with IL-1β is beneficial and useful for enhancing the production of porcine blastocysts in vitro.

This study investigated the effect of a co-culture of Scenedesmus dimorphus and nitrifiers using artificial wastewater on the removal of ammonium, nitrate and phosphate in the advanced treatment. To test the synergistic effect of the co-culture, we compared the co-culture treatment with the cultures using S. dimorphus-only and nitrifiers-only treatment as controls. After 6 days of incubation, nitrate was removed only in the co-culture treatment and total amount of N removal was 1.3 times and 1.6 times higher in the co-culture treatment compared to those in the S. dimorphus- and nitrifiers-only treatments, respectively. In case of total amount of P, co-culture treatment removed 1.2 times and 12 times more P than the S. dimorphus -and nitrifiers-only conditions, respectively. This indicates that the co-culture improved removal rates for ammonium, nitrate, and phosphate. This further implies that there was no need for denitrification of nitrate and luxury uptake of P processes because nitrate and phosphate can be removed from the uptake by S. dimorphus. In addition, co-culture condition maintained high DO above 7 mg/L without artificial aeration, which is enough for nitrification, implying that co-culture has a potential to decrease or remove aeration cost in the wastewater treatment plants.

Three-dimensional (3D) culture system is useful technique for study of in vivo environment and it was used various experiments. This study was investigated to establish of embryo co-culture system and changes of PAs activity in 3D cultured endometrial cells of pigs. In results, growth of stromal cells into gel matrix were detected only with endometrial and myometrial cells. The most rapid growth of stromal cells were confirmed in 2.5x105cells/ml and gel matrix containing 15% FBS. Expression of urokinase-PA (uPA) after treatment of hCG (0.5, 1.0, 1.5 and 2.0 IU/ml) were higher than without hCG, but, there are not significant difference among the treatment. On the other hand, expression of uPA after treatment of IL-1β (0.1, 1, 10 and 100 ng/ml) were higher than without IL-1β, but, there are not significant difference. Expression of uPA after treatment of estrogen (0.2, 2, 20 and 200 ng/ml) were not difference, but PA activity was significantly decreased (p＜0.05). Blastocyst was producing in PZM-3 medium containing FBS and endometrial cells were grown in PZM-3 medium. When embryos development with cultured endometrial cells, cleavage rates were not significant difference and blastocyst were not produced in co-culture with stromal cells and 3D culture system. 3D culture system had similar activity to in vivo tissue and these features are very useful for study of in vivo physiology. Nevertheless 3D culture system was not proper in embryo co-culture system. Therefore, we suggest that 3D culture system with embryo co-culture need continuous research.

Recent 2 decades, including in vitro maturation (IVM), assisted reproductive technologies (ARTs) achieved noteworthy development. However the efficiency of ARTs with in vitro matured oocytes is still lower than that with in vivo oocytes. To overcome those limitations, many researchers attempted to adapt co-culture system during IVM and consequently maturation efficiency has been increased. The beneficial effects of applying co-culture system is contemplated base on communication and interaction between various somatic cells and oocytes, achievement of paracrine factors, and spatial effects of extracellular matrix (ECM) from somatic cell surface. The understanding of co-culture system can provide some information to narrow the gap between in vitro and in vivo. Here we will review current studies about issues for understanding cu-culture system with various somatic cells to improve in vitro maturation microenvironment and provide bird view and strategies for further studies.

This study examined the motility of either the unattached(upper) or attached(lower) Hanwoo sperm to bovine oviduct epithelial cell(BOEC) monolayers to determine whether there are any changes in their motility during co-culture. The cleavage and blastocyst development rate were compared among different preincubation methods in-vitro, after oocytes were fertilized in-vitro with Hanwoo sperm on BOEC monolayers. The motility of frozen-thawed sperm in BOEC co-culture group was significantly higher than controls, especially at 5 hours and 6 hours (p<0.05) of incubation, in sperm treatment medium without heparin and caffeine. The motility of frozen-thawed sperm in BOEC co-culture group was significantly higher than controls, especially at 3 hours (p<0.05) and 6 hours (p<0.01), in sperm treatment medium containing heparin and caffeine. The motility of the attached( lower) sperm was significantly higher than the unattached(upper) sperm during co-culture with BOEC at all times(p<0.01 or p<0.05), except for 6 hours. After Hanwoo oocytes were fertilized in-vitro with the sperm that had been co-cultured with BOEC in sperm treatment medium containing heparin and caffeine, we determined the cleavage and blastocyst development rate, according to the preincubation methods. Both the cleavage and blastocyst development rate from 2 hour preincubation group were the highest, but significant difference was not recognized. These results show that BOEC plays an important role on sperm hyperactivation related to capacitation regardless of heparin and caffeine in sperm treatment medium. However, oviduct epithelial cell had no significant effect on the development of embryos after in-vitro fertilization in the presence of added heparin and caffeine in sperm treatment medium.

The objective of this study was to evaluate the effects of co-culture of bovine oocytes with cumulus cells on in vitro maturation and development following in vitro fertilization in bovine oocytes. Bovine cumulus-oocyte complexes (COCs) and denuded oocytes (DO) were co-cultured with the cumulus cells in TCM199 for 20~22 hr, and evaluated the nuclear type of oocyte. After in vitro maturation, oocytes were coincubated for in vitro fertilization with frozen-thawed spermatozoa selected by 65% percoll in DM-Heparin and DM-Caffeine for 15~18 hr. Presumptive zygotes were cultured for 48 hr in CR1aa in vitro culture medium with 10% FBS, and evaluated the cleavage rates. The results confirmed that the highest percentage of metaphase II (M-II) stage was observed in COCs (30.1±3.5%, 24.2±1.8%) as compared to DO (7.1±1.3%, 17.4±13.9%) (p<0.05). In addition, the increased cleavage rates were obtained from COCs (69.6±2.1%, 75.6±2.9%) when compared to DO (21.6±7.5%, 29.5±12.6%) (p<0.05). In conclusion, this study suggested that cumulus cells secreted positive factors during in vitro maturation of oocytes and early embryonic development after in vitro fertilization of bovine oocytes.

Somatic cells such as oviduct epithelial cell, uterine epithelial cell, cumulus-granulosa cell and buffalo rat river cell has been used to establish an effective culture system for bovine embryos produced in in vitro. But nitric oxide (NO) metabolites secreted from somatic cells were largely arrested the development of bovine in vitro matured/ in vitro fertilized (IVM/IVF) embryos, suggesting that NO was induced the embryonic toxic substance into culture medium. The objective of this study was to investigate whether BOEC co-culture system can ameliorate the NO-mediated oxidative stress in the culture of bovine IVM/IVF embryos. Therefore, we evaluated the developmental rate of bovine IVM/IVF embryos under BOEC co-culture system in the presence or absence of sodium nitroprusside (SNP), as a NO donor, and also detected the expression of growth factor (TGF-p , EGF and IGFBP) and apoptosis (Caspase-3, Bax and Bcl-2) genes. The supplement of SNP over 5 uM was strongly inhibited blastocyst development of bovine IVM/IVF embryos than in control and 1 uM SNP group (Table 2). The developmental rates beyond morulae stages of bovine IVM/IVF embryos co-cultured with BOEC regardless of SNP supplement (40.4% in 5 uM SNP+ BOEC group and 65.1% in BOEC group) were significantly increased than those of control (35.0%) and SNP single treatment group (23.3%, p<0.05: Table 3). The transcripts of Bax and Caspase-3 genes were detected in all experiment groups (1:Isolated fresh cell (IFC), 2:Primary culture cell (PCC), 3:PCC after using the embryo culture, 4: PCC containing 5 uM SNP and 5: PCC containing 5 uM SNP after using the embryo culture), but Bcl-2 gene was not detected in IFC and PCC (Fig. 1). In the expression of growth factor genes, TGF-p gene was found in all experimental groups, and EGF and IGFBP genes were not found in IFC and PCC (Fig. 2). These results indicate that BOEC co-culture system can increase the development beyond morula stages of bovine IVM/IVF embryos, possibly suggesting the alleviation of embryonic toxic substance like nitric oxide.

In vitro maturation of porcine immature cumulus-enclosed oocytes can be enhanced by co-incubation with spermatozoa even before fertilization. The aim of this study was to determine whether the addition of spermatozoa into the culture medium can stimulate the meiosis resumption of porcine cumulus-enclosed oocytes arrested at germinal vesicle (GV). Cumulus-enclosed oocytes (CEOs) were collected from follicles of 3 to 5mm diameter. Porcine CEOs were cultured in tissue culture medium containing various meiosis inhibitors and spermatozoa. Oocytes were examined for evidence of GV and GV breakdown after 24 h culture. After 24 h culture 43.8% of oocytes cultured in only TCM 199 remained at GV stage whereas 56.2% of oocytes were able to resume meiosis. When porcine CEOs were cultured in the medium with meiosis inhibitor such as, dibutyryl cAMP (dbcAMP) and forskolin (Fo), more than 90% of oocytes were not able to resume meiosis. However, co-culture of porcine CEOs with spermatozoa was able to overcome the inhibitory effect of dbcAMP and Fo. Irrespective of the presence of 3-isobutyl-1-methylxanthine (IBMX), no difference was observed in the proportion of oocyte reached germinal vesicle breakdown (GVBD). The present study suggests that dbcAMP and Fo prevent the spontaneous maturation of competent oocyte in culture after isolation from follicles and that mammalian spermatozoa contain a substance(s) that improves meiosis resumption in vitro of porcine cumulus-enclosed oocytes.