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Rat Primary Hepatocyte의 2차원 배양과 3차원 배양에 따른 생리 활성능과 대사능에 관한 연구 KCI 등재
Evaluation of primary hepatocyte function using 2D or 3D culture method for primary rat hepatocytes
pp.169-177
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.
재료 및 방법
1. 시약 및 배양액
2. 간세포 분리
3. 간세포의 배양
4. 면역 형광 염색
5. Dexamethasone(Dex)을 이용한 CYP3A1 유도
6. 유전자 정량분석(Real-time PCR)
7. 통계 처리
결 과
1. 랫트 간으로부터 얻어낸 Hepatocyte의 형태학적 분석
2. 면역 형광 염색에 의한 간세포 검증
3. 체외 배양 조건에 따른 일차 간세포의 생리학적 특성 분석
4. 체외 배양 조건에 따른 일차 간세포의 생존능
5. 체외에서 배양된 일차 간세포에서 CYP3A1 발현
고 찰
REFERENCES
