Creating rat hepatocyte organoid as an in vitro model for drug testing

• 作者: Bing-Qi Zhang Hong Bu Ji Bao Li Li Qiong Wu Sheng-Fu Li Xing-Long Zhu Ya-Na Qi Yan-Yan Zhou Yi Li Yu-Ting He Yun-Lin Zhang
• 来源期刊: 世界干细胞杂志:英文版(电子版)
• 学科: 医学
• 关键词: Organoid Primary hepatocytes Stem cells Liver therapies Extracellular matrix Drug screening
• 年，卷（期）: 2020,（10）
• 页码范围: 1184-1195
• 页数: 12页
• 分类号: R57

摘要

BACKGROUND Liver organoids have recently been applied as models for liver disease and drug screening,especially when combined with liver-on-a-chip technologies.Compared to hepatocyte-like cells,primary hepatocytes have high functionality but cannot maintain their function when cultured in vitro.Mesenchymal stem cells(MSCs)enhance hepatocyte function and maintain hepatocyte metabolism when co-cultured with hepatocytes.MSCs can help induced pluripotent stem cells to generate an organoid structure via the MSC-based traction force triggered by extracellular matrix(ECM)proteins.In this study,primary hepatocytes were cocultured with MSCs on a liver-derived ECM to generate liver organoids within a short duration.AIM To create hepatocyte organoids by co-culturing primary hepatocytes with MSCs on a porcine liver extracellular matrix(PLECM)gel.METHODS Perfusion and enzymatic hydrolysis were used to form the PLECM gel.Rat hepatocytes and human MSCs were mixed and plated on pre-solidified PLECM gel in a 48-well plate for 48 h to generate organoids.Generated organoids were evaluated through hematoxylin and eosin,periodic acid-Schiff,immunohistological,and immunofluorescence staining,and quantitative PCR for alb,CYP450 gene markers,and urea cycle genes.Culture medium was collected to detect albumin(ALB)and urea production on days 2,4,6,8,14,and 20.RESULTS The whole porcine liver was perfused and enzymatically hydrolyzed to form a PLECM gel.The structural components and basement membrane composition of the ECM,such as collagen type I,collagen type IV,fibronectin,and laminin,were demonstrated to be retained.Through interaction of human MSCs with the liverderived ECM,primary hepatocytes and human MSCs assembled together into a 3D construction and generated primary hepatocyte organoids for 48 h.The mRNAs of the gene alb,the CYP450 gene markers cyp1a1,cyp1a2,and cyp3a2 as well as urea cycle genes arg-1,asl,ass-1,cps-1,nags were highly expressed in hepatocyte organoids.Long-term survival of the primary hepatocyte organoids,as well a