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Hum Gene Ther Methods. 2015 Dec;26(6):197-210. doi: 10.1089/hgtb.2015.080. Epub 2015 Nov 10.

Use of a Closed Culture System to Improve the Safety of Lentiviral Vector Production.

Wu T1,2,3, Bour G4, Durand S1,2, Lindner V5,6, Gossé F1,2, Zona L1,2, Certoux JM7, Diana M4,8, Baumert TF1,2,8,9, Marescaux J4,8,9, Mutter D4,8,9, Pessaux P1,2,8,9, Robinet E1,2,8.

Author information

1
1 INSERM , UMR 1110, Strasbourg, France.
2
2 Université de Strasbourg , Strasbourg, France.
3
3 Department of Hepatobiliary and Pancreatic Surgery, Second Affiliated Hospital of Kunming Medical University Kunming , Yunnan, People's Republic of China.
4
4 IRCAD, Research Institute Against Digestive Cancer , Strasbourg, France.
5
5 Centre de Ressources Biologiques, Hôpitaux Universitaires de Strasbourg , Strasbourg, France.
6
6 Département de Pathologie, Hôpitaux Universitaires de Strasbourg , Strasbourg, France.
7
7 Etablissement Français du Sang Bourgogne/Franche-Comté , Besançon, France.
8
8 IHU Strasbourg, Institute for Image-Guided Surgery , Strasbourg, France.
9
9 Pôle Hépatodigestif, Nouvel Hôpital Civil, Hôpitaux Universitaires de Strasbourg , Strasbourg, France .

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.

PMID:
26467420
DOI:
10.1089/hgtb.2015.080
[Indexed for MEDLINE]

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