Hiller Thomas, Röhrs Viola, Dehne Eva-Maria, Wagner Anke, Fechner Henry, Lauster Roland, Kurreck Jens
Department of Applied Biochemistry, Institute of Biotechnology, Berlin University of Technology.
Department of Medical Biotechnology, Institute of Biotechnology, Berlin University of Technology.
J Vis Exp. 2016 Oct 24(116):54633. doi: 10.3791/54633.
This protocol describes the generation of a three-dimensional (3D) ex vivo liver model and its application to the study and development of viral vector systems. The model is obtained by repopulating the extracellular matrix of a decellularized rat liver with a human hepatocyte cell line. The model permits studies in a vascularized 3D cell system, replacing potentially harmful experiments with living animals. Another advantage is the humanized nature of the model, which is closer to human physiology than animal models. In this study, we demonstrate the transduction of this liver model with a viral vector derived from adeno-associated viruses (AAV vector). The perfusion circuit that supplies the 3D liver model with media provides an easy means to apply the vector. The system permits monitoring of the major metabolic parameters of the liver. For final analysis, tissue samples can be taken to determine the extent of recellularization by histological techniques. Distribution of the virus vector and expression of the delivered transgene can be analyzed by quantitative PCR (qPCR), Western blotting and immunohistochemistry. Numerous applications of the vector model in basic research and in the development of gene therapeutic applications can be envisioned, including the development of novel antiviral therapeutics, cancer research, and the study of viral vectors and their potential side effects.
本方案描述了三维（3D）离体肝脏模型的构建及其在病毒载体系统研究与开发中的应用。该模型通过用人肝细胞系重新填充去细胞大鼠肝脏的细胞外基质获得。该模型允许在血管化的3D细胞系统中进行研究，取代对活体动物可能有害的实验。另一个优点是该模型的人源化特性，它比动物模型更接近人类生理学。在本研究中，我们展示了用源自腺相关病毒的病毒载体（AAV载体）转导该肝脏模型。为3D肝脏模型提供培养基的灌注回路提供了一种应用载体的简便方法。该系统允许监测肝脏的主要代谢参数。为进行最终分析，可采集组织样本，通过组织学技术确定再细胞化程度。病毒载体的分布和所递送转基因的表达可通过定量PCR（qPCR）、蛋白质印迹法和免疫组织化学进行分析。可以设想该载体模型在基础研究和基因治疗应用开发中的众多应用，包括新型抗病毒疗法的开发、癌症研究以及病毒载体及其潜在副作用的研究。
