Cationic lipid-mediated transfection of differentiated Caco-2 cells: a filter culture model of gene delivery to a polarized epithelium: Pharm Res

A. N. Uduehi, S. H. Moss, J. Nuttall, C. W. Pouton

Research output: Contribution to journalArticleResearchpeer-review

Abstract

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.
Original languageEnglish
Pages (from-to)1805-1811
Number of pages7
JournalPharmaceutical Research
Volume16
Publication statusPublished - 1999

Keywords

  • Caco-2 Cells Cations Cell Differentiation/drug effects Cells, Cultured DNA/genetics *Drug Carriers Epithelial Cells/*physiology Filtration Humans Lipids/*chemistry Liposomes Particle Size Plasmids/genetics Taurocholic Acid/metabolism Transfection/*methods

Cite this

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title = "Cationic lipid-mediated transfection of differentiated Caco-2 cells: a filter culture model of gene delivery to a polarized epithelium: Pharm Res",
abstract = "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.",
keywords = "Caco-2 Cells Cations Cell Differentiation/drug effects Cells, Cultured DNA/genetics *Drug Carriers Epithelial Cells/*physiology Filtration Humans Lipids/*chemistry Liposomes Particle Size Plasmids/genetics Taurocholic Acid/metabolism Transfection/*methods",
author = "Uduehi, {A. N.} and Moss, {S. H.} and J. Nuttall and Pouton, {C. W.}",
note = "M1 - 12 Uduehi, A N Moss, S H Nuttall, J Pouton, C W eng Research Support, Non-U.S. Gov't 2000/01/22 09:00 Pharm Res. 1999 Dec;16(12):1805-11.",
year = "1999",
language = "English",
volume = "16",
pages = "1805--1811",
journal = "Pharmaceutical Research",
issn = "0724-8741",
publisher = "American Association of Pharmaceutical Scientists",

}

Cationic lipid-mediated transfection of differentiated Caco-2 cells: a filter culture model of gene delivery to a polarized epithelium : Pharm Res. / Uduehi, A. N.; Moss, S. H.; Nuttall, J.; Pouton, C. W.

In: Pharmaceutical Research, Vol. 16, 1999, p. 1805-1811.

Research output: Contribution to journalArticleResearchpeer-review

TY - JOUR

T1 - Cationic lipid-mediated transfection of differentiated Caco-2 cells: a filter culture model of gene delivery to a polarized epithelium

T2 - Pharm Res

AU - Uduehi, A. N.

AU - Moss, S. H.

AU - Nuttall, J.

AU - Pouton, C. W.

N1 - M1 - 12 Uduehi, A N Moss, S H Nuttall, J Pouton, C W eng Research Support, Non-U.S. Gov't 2000/01/22 09:00 Pharm Res. 1999 Dec;16(12):1805-11.

PY - 1999

Y1 - 1999

N2 - 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.

AB - 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.

KW - Caco-2 Cells Cations Cell Differentiation/drug effects Cells, Cultured DNA/genetics Drug Carriers Epithelial Cells/physiology Filtration Humans Lipids/chemistry Liposomes Particle Size Plasmids/genetics Taurocholic Acid/metabolism Transfection/methods

M3 - Article

VL - 16

SP - 1805

EP - 1811

JO - Pharmaceutical Research

JF - Pharmaceutical Research

SN - 0724-8741

ER -