title: Use of a Closed Culture System to Improve the Safety of Lentiviral Vector Production.
abstract: We evaluated the possibility of introducing a combination of six oncogenes into primary porcine hepatocytes (PPH) using a lentiviral vector (LV)-mediated gene transfer in order to develop a porcine hepatocellular carcinoma model based on autologous transplantation of ex vivo-transformed hepatocytes. The six oncogenes were introduced into three plasmids, hence enabling the production of LVs encoding a luciferase reporter gene and hTERT+p53(DD), cyclinD1+CDK4(R24C), and c-myc(T58A)+HRas(G21V) genes, respectively. In order to improve the protection of the laboratory personnel manipulating such LVs, we used a compact cell culture cassette (CliniCell(®) device) as a closed cell culture system. We demonstrated that the CliniCell device allows to produce LVs, through plasmid transfection of 293T cells, and, after transfer to a second cassette, to transduce PPH with a similar efficacy as conventional open cell culture systems such as flasks or Petri dishes. Additionally, it is possible to cryopreserve at -80°C the transduced cells, directly in the CliniCell device used for the transduction. In conclusion, the use of a closed culture system for the safe handling of oncogene-encoding LVs lays the foundation for the development of porcine tumor models based on the autologous transplantation of ex vivo-transformed primary cells.
authors.affiliation: 1 INSERM , UMR 1110, Strasbourg, France.|2 Université de Strasbourg , Strasbourg, France.|3 Department of Hepatobiliary and Pancreatic Surgery, Second Affiliated Hospital of Kunming Medical University Kunming , Yunnan, People's Republic of China.
authors.forename: Tao
authors.initials: T
authors.lastname: Wu
authors.affiliation: 4 IRCAD, Research Institute Against Digestive Cancer , Strasbourg, France.
authors.forename: Gaëtan
authors.initials: G
authors.lastname: Bour
authors.affiliation: 1 INSERM , UMR 1110, Strasbourg, France.|2 Université de Strasbourg , Strasbourg, France.
authors.forename: Sarah
authors.initials: S
authors.lastname: Durand
authors.affiliation: 5 Centre de Ressources Biologiques, Hôpitaux Universitaires de Strasbourg , Strasbourg, France.|6 Département de Pathologie, Hôpitaux Universitaires de Strasbourg , Strasbourg, France.
authors.forename: Véronique
authors.initials: V
authors.lastname: Lindner
authors.affiliation: 1 INSERM , UMR 1110, Strasbourg, France.|2 Université de Strasbourg , Strasbourg, France.
authors.forename: Francine
authors.initials: F
authors.lastname: Gossé
authors.affiliation: 1 INSERM , UMR 1110, Strasbourg, France.|2 Université de Strasbourg , Strasbourg, France.
authors.forename: Laetitia
authors.initials: L
authors.lastname: Zona
authors.affiliation: 7 Etablissement Français du Sang Bourgogne/Franche-Comté , Besançon, France.
authors.forename: Jean-Marie
authors.initials: JM
authors.lastname: Certoux
authors.affiliation: 4 IRCAD, Research Institute Against Digestive Cancer , Strasbourg, France.|8 IHU Strasbourg, Institute for Image-Guided Surgery , Strasbourg, France.
authors.forename: Michele
authors.initials: M
authors.lastname: Diana
authors.affiliation: 1 INSERM , UMR 1110, Strasbourg, France.|2 Université de Strasbourg , Strasbourg, France.|8 IHU Strasbourg, Institute for Image-Guided Surgery , Strasbourg, France.|9 Pôle Hépatodigestif, Nouvel Hôpital Civil, Hôpitaux Universitaires de Strasbourg , Strasbourg, France .
authors.forename: Thomas F
authors.initials: TF
authors.lastname: Baumert
authors.affiliation: 4 IRCAD, Research Institute Against Digestive Cancer , Strasbourg, France.|8 IHU Strasbourg, Institute for Image-Guided Surgery , Strasbourg, France.|9 Pôle Hépatodigestif, Nouvel Hôpital Civil, Hôpitaux Universitaires de Strasbourg , Strasbourg, France .
authors.forename: Jacques
authors.initials: J
authors.lastname: Marescaux
authors.affiliation: 4 IRCAD, Research Institute Against Digestive Cancer , Strasbourg, France.|8 IHU Strasbourg, Institute for Image-Guided Surgery , Strasbourg, France.|9 Pôle Hépatodigestif, Nouvel Hôpital Civil, Hôpitaux Universitaires de Strasbourg , Strasbourg, France .
authors.forename: Didier
authors.initials: D
authors.lastname: Mutter
authors.affiliation: 1 INSERM , UMR 1110, Strasbourg, France.|2 Université de Strasbourg , Strasbourg, France.|8 IHU Strasbourg, Institute for Image-Guided Surgery , Strasbourg, France.|9 Pôle Hépatodigestif, Nouvel Hôpital Civil, Hôpitaux Universitaires de Strasbourg , Strasbourg, France .
authors.forename: Patrick
authors.initials: P
authors.lastname: Pessaux
authors.affiliation: 1 INSERM , UMR 1110, Strasbourg, France.|2 Université de Strasbourg , Strasbourg, France.|8 IHU Strasbourg, Institute for Image-Guided Surgery , Strasbourg, France.
authors.forename: Eric
authors.initials: E
authors.lastname: Robinet
mesh_terms: D000818:Animals; D061249:Batch Cell Culture Techniques / Q000295:instrumentation / Q000379:methods / Q000592:standards; D015870:Gene Expression; D017930:Genes, Reporter; D005822:Genetic Vectors / Q000235:genetics* / Q000302:isolation & purification*; D057809:HEK293 Cells; D022781:Hepatocytes; D006801:Humans; D016086:Lentivirus / Q000235:genetics*; D013552:Swine; D014161:Transduction, Genetic; D019076:Transgenes; D014779:Virus Replication