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.

There is a growing interest in the application of primary hepatocytes for treatment of liver diseases in humans and for drug development. Several studies have focused on long-term survival and di-differentiation blocking of primary hepatocytes in an in vitro culture system. Therefore, the present study also aimed to optimize an in vitro culture system using primary rat hepatocytes. Primary rat hepatocytes from 6-week-old male Crl:CD rats were isolated using a modified two-step collagenase perfusion. Healthy 3.5 × 106 primary rat hepatocytes were seeded into a 2 dimensional (2D) culture in a 25T culture flask coated with collagen type I or into a 3D culture in a 125-ml spinner flask for 7 days. Production of plasma protein (ALB and TF), apoptosis (BAX and BCL2), and CYP (CYP3A1) related genes were compared between the 2D and 3D culture systems. The 3D culture system had an advantage over the 2D system because of the relatively high expression of ALB and low expression of BAX in the 3D system. However, the level of CYP3A1 did not improve in the 3D culture with and without the presence of a dexamethasone inducer. Therefore, 3D culture has an advantage for albumin production and primary rat hepatocyte survivability, but a low expression of CYP3A1 indicated that primary rat hepatocytes require a high–density culture for stress reduction by continuous flow.

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.

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.

Highly homogeneous and functional stem cell-derived hepatocyte-like cells (HLCs) are considered a promising option in the treatment of liver disease and the development of effective in vitro toxicity screening tool. However, the purity of cells and expression and/or activity of drug metabolizing enzymes in stem cell-derived HLCs are usually too low to be useful for clinical or in vitro applications. Here, we describe a highly optimized differentiation protocol, which produces more than 90% albumin-positive HLCs with no purification process. In addition, we show that hepatic enzyme gene expressions and activities were significantly improved by generating three-dimensional (3D) spheroidal aggregate of HLCs. The 3D differentiation method increased expressions of nuclear receptors that regulate the proper expression of key hepatic enzymes. Furthermore, a significantly increased hepatic functions such as albumin and urea secretion were observed in 3D hepatic spheroids and HLCs in the spheroid exhibited morphological and ultrastructural features of normal hepatocytes. Importantly, we show that repeated exposures to xenobiotics facilitated the functional maturation of HLC, as confirmed by increased expression of genes for drug metabolizing enzymes and transcription factors. In conclusion, the 3D culture system with repeated exposures to xenobiotics may be a new strategy for enhancing hepatic maturation of stem cell-derived HLCs as a cell source for in vitro high-throughput hepatotoxicity models.

Hepatocyte-like cells (HLCs) derived from human pluripotent stem cells have received extensive attention in the development of drug screening and toxicity testing. However, it has been reported that stem cell-derived HLCs showed hepatic functions that were too limited to be of use in drug screening and toxicity testing, possibly due to the lack of sufficient intercellular communication under conventional two-dimensional (2D) culture conditions. Therefore, a 3D differentiation system may overcome the in vitro limitation of 2D culture to produce stem cell-derived hepatocytes with mature metabolic functions. In this study, the feasibility of using a silicone-based spherofilm, specifically designed to produce spherical cell clusters, to generate uniformly sized 3D hepatic spheroids from hESCs was investigated. Hepatic spheroids generated on the spherofilm showed more homogenous size and shape than those generated in conventional low-attachment suspension culture dishes. Results of immunohistochemical analysis showed that expression of the mature hepatic marker albumin (ALB) increased over time during the hepatic maturation process. Furthermore, the 3D culture system mimicked the in vivo 3D microenvironment. Laminin, which is an important component of hepatic ECM, was expressed in hepatic spheroids. The results of immunohistochemical analysis indicated that the 3D culture environment is capable of generating an in vivo-like microenvironment. In addition, quantitative PCR analysis showed that the mature hepatic marker ALB and cytochrome P450 (CYP) enzymes CYP3A4 and CYP3A7 were expressed at higher levels in 3D culture than in 2D culture. This indicates that the 3D culture system is suitable for hepatic maturation and that our size-controlled 3D culture conditions might accelerate hepatic function. These results suggest that 3D hepatic spheroids significantly enhance metabolic maturation of hepatocytes derived from hESCs