Use of a Closed Culture System to Improve the Safety of Lentiviral Vector Production.
Fiche publication
Date publication
novembre 2015
Auteurs
Membres identifiés du Cancéropôle Est :
Pr BAUMERT Thomas, Dr LINDNER Véronique, Pr MARESCAUX Jacques, Pr PESSAUX Patrick
Tous les auteurs :
Wu T, Bour G, Durand S, Lindner V, Gosse F, Zona L, Certoux JM, Diana M, Baumert TF, Marescaux J, Mutter D, Pessaux P, Robinet E
Lien Pubmed
Résumé
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+p53DD, cyclinD1+CDK4R24C, and c-mycT58A+HRasG21V genes, respectively. In order to improve the protection of the laboratory personnel manipulating such LVs, we used a compact cell culture cassette (CliniCell(R) 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 degrees 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.
Référence
Hum Gene Ther Methods. 2015 Nov 10.