PURPOSE: The use of rapidly dividing in vitro cell culture systems to assess the efficiency of gene delivery is now recognised as a poor indicator of in vivo success. We investigated whether differentiated Caco-2 cell filter-cultures would make a more suitable model for studying gene transfer to an epithelium. METHODS: Caco-2 cells were cultured on semi-permeable membrane filters into differentiated polarised monolayers. Monolayer differentiation was assessed by monitoring the transport of taurocholic acid. Cells at different stages of differentiation were transfected with DNA/DOTAP lipoplexes and later analysed for reporter gene activity. The uptake of radiolabled DNA was also evaluated at various stages of differentiation. RESULTS: Caco-2 cultures developed a resistance to lipoplex-mediated transfection as early as three days, when some cells were still dividing and undifferentiated. As cultures matured, expression of reporter gene progressively decreased partly due to reduced internalisation of DNA. The resistance to transfection could be overcome in part by pre-treatment of monolayers with calcium chelating agents or surfactants. However, transgene expression in treated monolayers was still significantly lower than that in dividing cultures. CONCLUSIONS: Differentiated Caco-2 cells are a more appropriate model for gene-transfer studies to the intestinal epithelium because they demonstrate a resistance to transfection similar to that observed in vivo.
PURPOSE: The use of rapidly dividing in vitro cell culture systems to assess the efficiency of gene delivery is now recognised as a poor indicator of in vivo success. We investigated whether differentiated Caco-2 cell filter-cultures would make a more suitable model for studying gene transfer to an epithelium. METHODS: Caco-2 cells were cultured on semi-permeable membrane filters into differentiated polarised monolayers. Monolayer differentiation was assessed by monitoring the transport of taurocholic acid. Cells at different stages of differentiation were transfected with DNA/DOTAP lipoplexes and later analysed for reporter gene activity. The uptake of radiolabled DNA was also evaluated at various stages of differentiation. RESULTS: Caco-2 cultures developed a resistance to lipoplex-mediated transfection as early as three days, when some cells were still dividing and undifferentiated. As cultures matured, expression of reporter gene progressively decreased partly due to reduced internalisation of DNA. The resistance to transfection could be overcome in part by pre-treatment of monolayers with calcium chelating agents or surfactants. However, transgene expression in treated monolayers was still significantly lower than that in dividing cultures. CONCLUSIONS: Differentiated Caco-2 cells are a more appropriate model for gene-transfer studies to the intestinal epithelium because they demonstrate a resistance to transfection similar to that observed in vivo.
Authors: D M Woodcock; M E Linsenmeyer; G Chojnowski; A B Kriegler; V Nink; L K Webster; W H Sawyer Journal: Br J Cancer Date: 1992-07 Impact factor: 7.640
Authors: Verena Hiebl; Daniel Schachner; Angela Ladurner; Elke H Heiss; Herbert Stangl; Verena M Dirsch Journal: Biol Proced Online Date: 2020-04-11 Impact factor: 3.244