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

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

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Batch Cell Culture Techniques* / instrumentation
  • Batch Cell Culture Techniques* / methods
  • Batch Cell Culture Techniques* / standards
  • Gene Expression
  • Genes, Reporter
  • Genetic Vectors / genetics*
  • Genetic Vectors / isolation & purification*
  • HEK293 Cells
  • Hepatocytes
  • Humans
  • Lentivirus / genetics*
  • Swine
  • Transduction, Genetic
  • Transgenes
  • Virus Replication*