title: Use of a three-dimensional humanized liver model for the study of viral gene vectors.
abstract: Reconstituted three-dimensional (3D) liver models obtained by engrafting hepatic cells into an extracellular matrix (ECM) are valuable tools to study tissue regeneration, drug action and toxicology ex vivo. The aim of the present study was to establish a system for the functional investigation of a viral vector in a 3D liver model composed of human HepG2 cells on a rat ECM. An adeno-associated viral (AAV) vector expressing the Emerald green fluorescent protein (EmGFP) and a short hairpin RNA (shRNA) directed against human cyclophilin b (hCycB) was injected into the portal vein of 3D liver models. Application of the vector did not exert toxic effects, as shown by analysis of metabolic parameters. Six days after transduction, fluorescence microscopy analysis of EmGFP production revealed widespread distribution of the AAV vectors. After optimization of the recellularization and transduction conditions, averages of 55 and 90 internalized vector genomes per cell in two replicates of the liver model were achieved, as determined by quantitative PCR analysis. Functionality of the AAV vector was confirmed by efficient shRNA-mediated knockdown of hCycB by 70-90%. Our study provides a proof-of-concept that a recellularized biological ECM provides a valuable model to study viral vectors ex vivo.
authors.affiliation: Department of Applied Biochemistry, Institute of Biotechnology, Berlin University of Technology, Berlin, Germany.
authors.forename: Anke
authors.initials: A
authors.lastname: Wagner
authors.affiliation: Department of Applied Biochemistry, Institute of Biotechnology, Berlin University of Technology, Berlin, Germany.
authors.forename: Viola
authors.initials: V
authors.lastname: Röhrs
authors.affiliation: Department of Medical Biotechnology, Institute of Biotechnology, Berlin University of Technology, Berlin, Germany.
authors.forename: Eva-Maria
authors.initials: EM
authors.lastname: Materne
authors.affiliation: Department of Applied Biochemistry, Institute of Biotechnology, Berlin University of Technology, Berlin, Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: Hiller
authors.affiliation: Department of Applied Biochemistry, Institute of Biotechnology, Berlin University of Technology, Berlin, Germany.
authors.forename: Radoslaw
authors.initials: R
authors.lastname: Kedzierski
authors.affiliation: Department of Applied Biochemistry, Institute of Biotechnology, Berlin University of Technology, Berlin, Germany.
authors.forename: Henry
authors.initials: H
authors.lastname: Fechner
authors.affiliation: Department of Medical Biotechnology, Institute of Biotechnology, Berlin University of Technology, Berlin, Germany.
authors.forename: Roland
authors.initials: R
authors.lastname: Lauster
authors.affiliation: Department of Applied Biochemistry, Institute of Biotechnology, Berlin University of Technology, Berlin, Germany. Electronic address: jens.kurreck@tu-berlin.de.
authors.forename: Jens
authors.initials: J
authors.lastname: Kurreck
mesh_terms: D000818:Animals; D021983:Cyclophilins / Q000235:genetics; D000229:Dependovirus / Q000235:genetics*; D005109:Extracellular Matrix; D055785:Gene Knockdown Techniques; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D056945:Hep G2 Cells; D006801:Humans; D008099:Liver / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D034741:RNA, Small Interfering / Q000235:genetics; D051381:Rats; D014161:Transduction, Genetic
chemical_list: D034741:RNA, Small Interfering; C081786:cyclophilin B; D049452:Green Fluorescent Proteins; D021983:Cyclophilins
keywords: Adeno-associated virus vectors; Extracellular matrix; Gene silencing; HepG2; Short hairpin RNA; Transduction