Study of Viral Vectors in a Three-dimensional Liver Model Repopulated with the Human Hepatocellular Carcinoma Cell Line HepG2.
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.
Department of Applied Biochemistry, Institute of Biotechnology, Berlin University of Technology.
Thomas
T
Hiller
Department of Applied Biochemistry, Institute of Biotechnology, Berlin University of Technology.
Viola
V
Röhrs
Department of Medical Biotechnology, Institute of Biotechnology, Berlin University of Technology.
Eva-Maria
EM
Dehne
Department of Applied Biochemistry, Institute of Biotechnology, Berlin University of Technology; Department of Bioprocess Engineering, Institute of Biotechnology, Berlin University of Technology.
Anke
A
Wagner
Department of Applied Biochemistry, Institute of Biotechnology, Berlin University of Technology.
Henry
H
Fechner
Department of Medical Biotechnology, Institute of Biotechnology, Berlin University of Technology.
Roland
R
Lauster
Department of Applied Biochemistry, Institute of Biotechnology, Berlin University of Technology; jens.kurreck@tu-berlin.de.
Jens
J
Kurreck
D000818:Animals; D006528:Carcinoma, Hepatocellular; D002460:Cell Line; D000229:Dependovirus; D015316:Genetic Therapy; D005822:Genetic Vectors; D006801:Humans; D008099:Liver; D008113:Liver Neoplasms; D051381:Rats; D014161:Transduction, Genetic; D019076:Transgenes