Creating rat hepatocyte organoid as an in vitro model for drug testing.
Creating rat hepatocyte organoid as an in vitro model for drug testing.
复制标题
创建大鼠肝细胞类器官作为药物测试的体外模型。
DOI：
10.4252/wjsc.v12.i10.1184
复制
发表时间：
2020-10-26
影响因子：
4.1
通讯作者：
Bu H
中科院分区：
文献类型：
--
作者：
He YT;Zhu XL;Li SF;Zhang BQ;Li Y;Wu Q;Zhang YL;Zhou YY;Li L;Qi YN;Bao J;Bu H
肝脏类器官最近已被用作肝脏疾病和药物筛选的模型，特别是当与肝脏芯片技术相结合时。与肝细胞样细胞相比，原代肝细胞具有高功能性，但在体外培养时不能维持其功能。间充质干细胞（MSC）与肝细胞共培养时，可增强肝细胞功能并维持肝细胞代谢。MSC可以通过细胞外基质（ECM）蛋白触发的基于MSC的牵引力帮助诱导多能干细胞生成类器官结构。在这项研究中，原代肝细胞与MSC在肝源性ECM上共培养，以在短时间内产生肝类器官。通过在猪肝脏细胞外基质（PLECM）凝胶上共培养原代肝细胞与MSC来创建肝细胞类器官。 灌注和酶水解用于形成PLECM凝胶。将大鼠肝细胞和人MSC混合并在48孔板中的预固化PLECM凝胶上铺板48小时以产生类器官。通过苏木精和伊红、高碘酸-希夫、免疫组织学和免疫荧光染色以及定量PCR对生成的类器官进行评估，以检测alb、CYP 450基因标志物和尿素循环基因。在第2、4、6、8、14和20天收集培养基以检测白蛋白（ALB）和尿素产生。将整个猪肝灌注并酶解以形成PLECM凝胶。ECM的结构成分和基底膜组成，如I型胶原、IV型胶原、纤连蛋白和层粘连蛋白，被证明被保留。通过人MSC与肝源性ECM的相互作用，原代肝细胞和人MSC组装在一起形成3D构建体，并产生原代肝细胞类器官48 h。基因alb、CYP 450基因标记物cyp 1a 1、cyp 1a 2和cyp 3a 2以及尿素循环基因arg-1、asl、ass-1、cps-1、nags的mRNA在肝细胞类器官中高度表达。原代肝细胞类器官的长期存活以及稳定的功能性通过体外ALB和尿素产生来证明。我们通过在肝源性ECM水凝胶上共培养肝细胞与MSC来创建原代肝细胞类器官的新方法可用于开发肝病模型和药物筛选。
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 co-cultured with MSCs on a liver-derived ECM to generate liver organoids within a short duration. To create hepatocyte organoids by co-culturing primary hepatocytes with MSCs on a porcine liver extracellular matrix (PLECM) gel. 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, immuno-histological, 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. 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 liver-derived 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 as stable functionality, was demonstrated via ALB and urea production in vitro. Our new method of creating primary hepatocyte organoids by co-culturing hepatocytes with MSCs on liver-derived ECM hydrogels could be used to develop models for liver disease and for drug screening.
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影响因子：
46.9
作者：
Azuma, Hisaya;Paulk, Nicole;Grompe, Markus
通讯作者：
Grompe, Markus
影响因子：
64.8
作者：
通讯作者：
--
影响因子：
5.9
作者：
Zhang RR;Koido M;Tadokoro T;Ouchi R;Matsuno T;Ueno Y;Sekine K;Takebe T;Taniguchi H
通讯作者：
Taniguchi H
影响因子：
14
作者：
Rennert, Knut;Steinborn, Sandra;Mosig, Alexander S.
通讯作者：
Mosig, Alexander S.
影响因子：
64.5
作者：
Sachs, Norman;de Ligt, Joep;Clevers, Hans
通讯作者：
Clevers, Hans