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.

Despite advancements in therapeutic approaches, radiotherapy and cisplatin-based chemotherapy remain primary noninvasive treatments for patients with oral squamous cell carcinoma (OSCC). Moreover, the 5-year survival rate for patients with OSCC has remained almost unchanged for several decades, and many side effects of chemotherapy still exist. In this study, three-dimensional (3D) models of OSCC were established using fibroblasts, and the efficacy of various biological inhibitors was evaluated. A culture of epithelial cells with two types of fibroblasts (hTERT-hNOFs and cancer-associated fibroblasts) within a type I collagen matrix resulted in the formation of a continuous layer of tightly packed cells compared to models without fibroblasts. Furthermore, the effects of biological chemicals, including Y27632, latrunculin A, and verteporfin, on these models were investigated. The stratified formation of the epithelial layer and invasion in OSCC 3D-culture models were effectively inhibited by verteporfin, whereas invasion was weakly inhibited by Y27632 and latrunculin. Collectively, the developed OSCC 3D-culture models established with fibroblasts demonstrated the potential for drug screening, with verteporfin showing promising efficacy.

아미노글리코사이드계 항생제(Aminoglycosides, AGs) 는 그람음성균과 양성균에 광범위하게 작용하는 동물용 의약품으로, 최근 배양육에 사용된다고 알려져 있어, 안 전성 관리를 위한 분석법 마련이 반드시 필요하다. AGs 는 고극성 화합물로 성분 간의 분리를 위해 이온쌍 시 약(ion-pairing reagent, IPR)을 사용하고 있으나 IPR을 이동상에 첨가하는 기존 분석방법의 경우 용매가 흐르 는 동안 질량분석기로 주입되는 IPR로 인해 기기적인 문 제가 발생할 가능성이 높아, IPR를 바이알에 직접 첨가 하는 분석방법을 검토하였다. 본 연구에서 10종 AGs 성 분에 대한 분석방법을 확인하고 유효성을 검증하였다. 검출한계와 정량한계는 각각 0.0001-0.0038 mg/kg 와 0.004-0.011 mg/kg의 범위로 나타났으며, 0.01-0.5 mg/ kg 범위 내의 직선성(R2)은 0.99 이상이었다. AGs의 시 료 회수율을 확인하고자 소고기와 세포배양배지(cell culture medium) 매질에서 회수율과 상대표준편차로 나 타낸 정밀도를 확인한 결과 각각 70.7-120.6% 및 0.2 to 24.7%로 나타났다. 기존의 이동상에 IP 첨가 방법과 비 교하였을 때 유사한 수준으로 양호하였다. 검증된 AGs 분석법은 국내 유통되는 닭고기, 소고기, 돼지고기 15품 목과 배양육 배지 첨가제 6품목에 적용해보았다. 그 결 과 국내 유통되는 육류 15품목 모두 AGs 성분이 검출 되지 않았으나, 세포배양배지에서 streptomycin은 695.85- 1152.71 mg/kg, dehydrostreptomyci은 6.35-11.11 mg/kg 로 검출되었다. 따라서 IRR을 바이알에 직접 첨가하는 LC-MS/MS 방식은 육류, 세포배양배지, 배지첨가제 중 AGs 분석 및 안전성 평가를 위한 기초자료로 활용될 것 으로 기대된다.

Background: Germ cells undergo towards male or female pathways to produce spermatozoa or oocyte respectively which is essential for sexual reproduction. Mesenchymal stem cells (MSCs) have the potential of trans-differentiation to the multiple cell lineages. Methods: Herein, rat MSCs were isolated from bone marrow and characterized by their morphological features, expression of MSC surface markers, and in vitro differentiation capability. Results: Thereafter, we induced these cells only by retinoic acid supplementation in MSC medium and, could able to show that bone marrow derived MSCs are capable to trans-differentiate into male germ cell-like cells in vitro. We characterized these cells by morphological changes, the expressions of germ cell specific markers by immunophenotyping and molecular biology tools. Further, we quantified these differentiated cells. Conclusions: This study suggests that only Retinoic acid in culture medium could induce bone marrow MSCs to differentiate germ cell-like cells in vitro . This basic method of germ cell generation might be helpful in the prospective applications of this technology.

In this study, when stem cell culture solution is used as a cosmetic ingredient, one of the most prominent problems is that the ingredients generally have low thermal stability. Therefore, in this study, in order to find out how the stem cell culture medium is heated or preserved at high temperature, the effect of various effects of stem cells on the various effects of the stem cells was investigated. Investigated. As a result of the experiment, the wound healing assay confirmed that the cell migration increased after 6 hours, and after 24 hours, it was confirmed that the cell mobility was increased and cell division was promoted, thereby being concentrated. As a result of investigating the amount of transdermal water loss by preparing a cosmetic product containing stem cell culture solution, it was confirmed that the culture solution addition group showed an improvement rate of 31% compared to the non-added group, thereby helping in skin wound recovery. As a result of this, it is considered that this point should be considered when the stem cell culture medium is used as an active ingredient in cosmetics in the future.

In the early development of parthenogenetic embryo, cytoplasm and nucleic acid fragmentation may be a cause of lower embryo development. The purpose of this study was to evaluate whether embryonic development and apoptosis factors can be reduced by controlling the in-vitro culture environment by the addition of hormones, pregnancy serum and uterine milk. Our study showed that the activity of Casp-3 increased within the cytoplasm when artificially used hormones to induce the incubation environment, and PCNA's manifestation was low. However, the addition of pregnant serum appeared to lower the Casp-3 activity compared to the other groups. In addition, MMP-9 activity was increased and early embryo development and cytoplasmic fidelity were also increased. Therefore, the results of the present study showed that the use of gestational serum in the development of parthenogenetic embryo inhibit apoptosis and increases cytoplasmic reorganization by natural environmental control in in vitro culture.

The establishment of porcine embryonic stem cells (ESCs) from porcine somatic cell nuclear transfer (SCNT) blastocysts is influenced by in vitro culture day of porcine reconstructed embryo and feeder cell type. Therefore, the objective of the present study was to determine the optimal in vitro culture period for reconstructed porcine SCNT embryos and mouse embryonic fibroblast (MEF) feeder cell type for enhancing colony formation efficiency from the inner cell mass (ICM) of porcine SCNT blastocysts and their outgrowth. As the results, porcine SCNT blastocysts produced through in vitro culture of the reconstructed embryos for 8 days showed significantly increased efficiency in the formation of colonies, compared to those for 7 days. Moreover, MEF feeder cells derived from outbred ICR mice showed numerically the highest efficiency of colony formation in blastocysts produced through in vitro culture of porcine SCNT embryos for 8 days and porcine ESCs with typical ESC morphology were maintained more successfully over Passage 2 on outbred ICR mice-derived MEF feeder cells than on MEF feeder cells derived from inbred C57BL/6 and hybrid B6CBAF1 mice. Overall, the harmonization of porcine SCNT blastocysts produced through in vitro culture of the reconstructed embryos for 8 days and MEF feeder cells derived from outbred ICR mice will greatly contribute to the successful establishment of ESCs derived from porcine SCNT blastocysts.

Fish ovarian germline stem cells (OGSCs) that have the abilities to self-renew and differentiate into functional gametes can be used in various researches and applications. A main issue to be solved for effective utilization of fish OGSCs is the development of their stable in vitro culture condition, but only few researches about fish OGSC culture have been reported so far. In this study, in order to find the clues to develop the culture condition for OGSCs from Japanese medaka (Oryzias latipes), we tried to establish somatic cell lines as a candidate for the feeder cells and evaluated its supporting effects on the culture of ovarian cell populations from O. latipes. As the results, the somatic cell lines could be established only from the embryonic tissues among three tissues derived from embryos, fins and ovaries. Three embryonic cell lines were tested as a feeder cell for the culture of ovarian cell population and all three cell lines induced cell aggregation formation of the cultured ovarian cells whereas the feeder-free condition did not. Furthermore, a significant cellular proliferation was observed in the ovarian cells cultured on two of three cell lines. As a trial to increase the capacity of the cell lines as a feeder cell that supports the proliferation of the cultured ovarian cells, we subsequently established a stable line that expresses the foreign O. latipes fibroblast growth factor 2 (FGF2) from an embryonic cell line and evaluated its effectiveness as a feeder cell. The ovarian cells cultured on FGF2 expressing feeder cells still formed cell aggregates but did not show a significant increase in cellular proliferation compared to those cultured on non-transformed feeder cells. The results from this study will provide the fundamental information for in vitro culture of medaka OGSCs.

Although somatic cell nuclear transfer (SCNT)-derived embryonic stem cells (ESCs) in pigs have great potential, their use is limited because the establishment efficiency of ESCs is extremely low. Accordingly, we tried to develop in-vitro culture system stimulating production of SCNT blastocysts with high performance in the colony formation and formation of colonies derived from SCNT blastocysts for enhancing production efficiency of porcine ESCs. For these, SCNT blastocysts produced in various types of embryo culture medium were cultured in different ESC culture medium and optimal culture medium was determined by comparing colony formation efficiency. As the results, ICM of porcine SCNT blastocysts produced through sequential culture of porcine SCNT embryos in the modified porcine zygote medium (PZM)-5 and the PZM-5F showed the best formation efficiency of colonies in α-MEM-based medium. In conclusion, appropriate combination of the embryo culture medium and ESC culture medium will greatly contribute to successful establishment of ESCs derived from SCNT embryos.

The osmolarity of a medium that is commonly used for in vitro culture (IVC) of oocytes and embryos is lower than that of oviductal fluid in pigs. In vivo oocytes and embryos can resist high osmolarities to some extent due to the presence of organic osmolytes such as glycine and alanine. These amino acids act as a protective shield to maintain the shape and viability in high osmotic environments. The aim of this study was to determine the effects of glycine or/and alanine in medium with two different osmolarities (280 and 320 mOsm) during IVC on embryonic development after parthenogenesis (PA) and somatic cell nuclear transfer (SCNT) in pigs. To this end, IVC was divided into two stages; the 0-2 and 3-7 days of IVC. In each stage, embryos were cultured in medium with 280, 320, or 360 mOsm and their combinations with or without glycine or/and alanine according to the experimental design. Treatment groups were termed as, for example, "T(osmolarity of a medium used in 0-2 days of IVC)-(osmolarity of a medium used in 3-7 days of IVC)" T280-280 was served as control. When PA embryos were cultured in medium with various osmolarities, T320-280 showed a significantly higher blastocyst formation (29.0%) than control (22.2%) and T360-360 groups (6.9%). Glycine treatment in T320-280 significantly increased blastocyst formation (50.4%) compared to T320-280 only (36.5%) while no synergistic was observed after treatment with glycine and alanine together in T320-280 (45.7%). In contrast to PA embryonic development, the stimulating effect by the culture in T320-280 was not observed in SCNT blastocyst development (27.6% and 23.7% in T280-280 and T320-280, respectively) whereas the number of inner cell mass cells was significantly increased in T320-280 (6.1 cells vs. 9.6 cells). Glycine treatment significantly improved blastocyst formation of SCNT embryos in both T280-280 (27.6% vs. 38.0%) and T320-280 (23.7% vs. 35.3%). Our results demonstrate that IVC in T320-280 and treatment with glycine improves blastocyst formation of PA and SCNT embryos in pigs.

In the last several decades, cell therapy research has increased worldwide. Many studies have been conducted on cell therapy, and have revealed that transplanted cells did not survive for long, and implanted cells remained inactive causing immune rejection depending on the patient’s condition. Therefore, studies on cell-free therapy need to be conducted. To overcome these limitations, an alternative is the use of supernatant from cells, called “conditioned media (CM).” During in vitro cell culture, culture media supply nutrients to maintain cell characteristics and viability. In the culture, cells not only consume nutrients but also release beneficial proteins and substances, which are called “secretome.” CM from cells can be stored for a long time and is easy to handle. Moreover, secretome in CM can also be measured; exact amount of secretome is important to set the standard value for disease treatment. Here, we reviewed studies on CM and confirmed that various secretomes from CM were identified in these studies. Moreover, these findings could benefit cell and animal studies in future. In conclusion, CM could be a potential candidate for an alternative to cell therapy.

The nature of molecular mechanisms governing embryo development is largely unknown, but recent reports have demonstrated that proper execution of programmed cell death is crucial for this process. The main objective of this study is to examine the mode of programmed cell death during nuclear transfer embryos development in porcine. In particular, the relative employment of two major pathways in programmed cell death; e.g. apoptosis (type I) and autophagy (type II) was compared. Oocytes use in the study was matured in vitro in the presence of 10% FBS maturation medium. After nuclear transfer embryos were cultured for each programmed cell death control factor [Cysteamine(Cyst : 0.4mM), 3-methyladenine(3MA : 2.5mM) and Rapamycin(RP : 100nM)] in TCM-199 medium supplemented with 0.1% BSA. In this study results of among the blastocysts development in 3MA; PCNA, MAP1LC3A and ATG5 RNA gene expression level increased in the order of IVF<Cyst < 3MA < RP. However Casp-3 and TNF-r RNA gene expression level decreased in the order of IVF < 3MA and RP< Cyst. The expression of mTOR showed a pattern opposite to that of MAP1LC3A. That is, its expression was the lowest in Cyst group. And next experiments analysis of MMP expression patterns. Analysed this MMPs enzyme activation to evaluate the effectiveness of high quality brastocyst culture in porcine. In this results of the enzymatic activity of MMP-2 and MMP-9 was assessed in culture, the level of active MMP-9 was higher expression in the medium of each 3MA and RP treatment group, with the level of another treatment group being relatively higher. These results suggest that the autophagy activation culture medium is more effective for stable and innovative nuclear transfer embryos development.

금(Au)나노물질은 광학 및 바이오 분야에서 다양하게 응용될 수 있을 뿐만 아니라 식품 농작물 재배에 무기비료제 로 사용 시 기능성 및 품질 향상에 긍정적인 영향을 줄 수 있는 것으로 알려지면서 금 나노입자를 이용한 건강기능식 품 또는 기능성 농작물 재배에 대한 연구개발이 진행되고 있다. 따라서 식품 농작물 잔류와 체내 흡수 가능성에 따른 식품 및 생체 내에서의 정확하고 정밀한 분석법 확립과 안전성검증이 요구된다. 본 연구에서는 유도결합플라즈마 질 량분석기(ICP-MS)를 이용한 금 나노입자의 정량 분석을 확립하고자 전처리 방법을 최적화하고, 회수율, 정확성 및 정 밀성을 분석하여Au의 분석법을 확립하였다. 확립된 Au 정량 분석법을 이용하여 생체시료 내에서 금 나노입자의 정량 분석을 수행하여 생체 매트릭스의 영향을 확인하였다. 이러한 분석법을 바탕으로 장내 micro fold (M) 세포를 모사한 follicle associated epithelium (FAE) 모델과 일반 장관 상피세포 조건을 모사한 Caco-2 monolayer 모델을 이용하여 금 나노입자의 유입량을 비교 연구하였다. 그 결과, 생체 매트릭스 존재에 따른 금 나노입자 정량분석에 미치는 영향은 없는 것으로 나타났으며, 금 나노입자는 장관 상피세포 내 M 세포에 의해 에너지 의존적 endocytosis 기작에 의해 체 내 유입될 수 있는 것으로 확인되었다. 이러한 식품 농작물 무기비료로서 금 나노입자의 식품 및 체내 분석법 확립연 구 및 체내 흡수기작 규명은 추후 안전성 평가에 기초자료로 활용될 수 있을 것이다.

전 세계적으로 원자력 발전소는 442기가 가동 중이며, 62기가 충원될 예정이다. 원자력 발전소의 증가에 따라 방사성 폐기물 유출에 대한 위험성도 증가하였다. 이러한 이유 때문 에, 방사성 폐기물의 처리는 인간, 동물, 식물을 포함하는 자 연 생태계를 보전하는 관점에서 중요하다. 또한, 방사성 폐 기물 유출은 그 지역뿐만 아니라 전 세계적으로 심각한 문 제를 야기한다. 본 연구는 입체 배양세포에 방사성 핵종원 소 (세슘, 스트론튬, 코발트)를 처리하였고 이에 대한 영향력 을 확인하였다. 입체 배양 구조체는 아가로오스 하이드로겔 을 이용하여 제작했으며 암세포 및 정상세포 (HeLa, HepG2, COS-7)를 사용하여 입체 배양을 실시 하였다. 입체 형태로 세포를 배양한 후 세슘, 스트론튬, 코발트 농도 변화에 따라 세포 생존능력을 분석하였다. 이때 입체 배양세포에서 생존 능력이 단층 배양세포 보다 최대 42% 우수한 것을 확인하였 다. 입체 배양구조체는 세포가 형태 및 생리학적으로 in vivo 환경인 조직과 비슷하게 배양을 가능하게 하였다. 따라서, 입체 배양구조체는 기존의 단층 배양 한계점인 in vivo 환경 에 적용시킬 수 없다는 한계를 극복하였다. 본 입체 배양 기 술이 중금속 독성평가 및 단시간 내에 다수의 물질 분석을 수행하는 고속 대량 스크리닝 기술에 활용될 것으로 기대한 다.

Spermatogenesis is initiated from spermatogonial stem cells (SSCs) that has an ability of self-renewal and unipotency to generate differentiating germ cells. The objective of this study is to develop the simple method for derivation of SSCs using non-sorting of both spermatogonia and feeder cells. Simply uncapsulated mouse testes were treated with enzymes followed by surgical mincing, and single cells were cultured in stempro-34TM cell culture media at 37℃. After 5 days of culture, aciniform of SSC colony was observed, and showed a strong alkaline phosphatase activity. Molecular characterization of mouse SSCs showed that most of the mouse SSC markers such as integrin α6 and β1, CD9 and Stra8. In addition, pluripotency embryonic stem cell (ESC) marker Oct4 were expressed, however Sox2 expression was lowered. Interestingly, expression of SSC markers such as Vasa, Dazl and PLZF were stronger than mouse ESC (mESC). This data suggest that generated mouse SSCs (mSSCs) in this study has at least similar biomarkers expression to mESC and mSSCs derived from other study. Immunocytochemistry using whole mSSC colony also confirmed that mSSCs generated from this study expressed SSC specific biomarkers such as c-kit, Thy1, Vasa and Dazl. In conclusion, mSSCs from 5 days old mouse testes were successfully established without sorting of spermatogonia, and this cells expressed both mESC and SSC specific biomarkers. This simple derivation method for mSSCs may facilitate the study of spermatogenesis.