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Three-dimensional cell-scaffold constructs promote efficient gene transfection: Implications for cell-based gene therapy
Periodical: Tissue Engineering ISBN: 1076-3279
Number: 5
Language: English
Pages: 585-598
Authors:Xie, Y. B., Yang, S. T., Kniss, D. A.
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Abstract
To date, introduction of gene-modified cells in vivo is still a critical limitation for cell-based gene therapy. In this study, based on tissue engineering techniques, we developed a three-dimensional (3-D) transfection system to be cell-based gene delivery vehicle. Human trophoblast-like ED27 and fibroblastic NIH3T3 cells were used as model cell lines. Cells were seeded onto PET fibrous matrices and plated on polyethylene terephathalate (PET) films as 2-D transfection control. The cell-matrices and cell-films were transfected with pCMV-beta gal and pEGFP (green fluorescent protein) reporter gene vectors using LipofectAmine(R) reagent. Gene expression on 3-D versus 2-D growth surface were investigated. The effects of seeding method, seeding density, porosity of the PET matrix, and culturing time of the cell-matrix complex on cDNA transfection and expression in the 3-D cell-matrix complex were also investigated. The beta -gal assay and GFP detection showed that 3-D transfection promoted a higher gene expression level and longer expression time as compared to 2-D transfection. There existed an optimal initial cell seeding density for gene transfection of 3-D cell-matrix complex. Cells seeded on PET matrices with a lower porosity (similar to 87%) had higher gene expression activities than cells in the matrices with a higher porosity (similar to 90%). Also, Higher gene expression levels of beta -gal were obtained for the more uniformly seeded matrices that were seeded with a depth-filtration method. The results from this study demonstrate the potential utility of cells seeded onto 3-D fibrous matrices as cell-based gene delivery vehicle for in vitro study of gene expression or in vivo gene therapy.
To date, introduction of gene-modified cells in vivo is still a critical limitation for cell-based gene therapy. In this study, based on tissue engineering techniques, we developed a three-dimensional (3-D) transfection system to be cell-based gene delivery vehicle. Human trophoblast-like ED27 and fibroblastic NIH3T3 cells were used as model cell lines. Cells were seeded onto PET fibrous matrices and plated on polyethylene terephathalate (PET) films as 2-D transfection control. The cell-matrices and cell-films were transfected with pCMV-beta gal and pEGFP (green fluorescent protein) reporter gene vectors using LipofectAmine(R) reagent. Gene expression on 3-D versus 2-D growth surface were investigated. The effects of seeding method, seeding density, porosity of the PET matrix, and culturing time of the cell-matrix complex on cDNA transfection and expression in the 3-D cell-matrix complex were also investigated. The beta -gal assay and GFP detection showed that 3-D transfection promoted a higher gene expression level and longer expression time as compared to 2-D transfection. There existed an optimal initial cell seeding density for gene transfection of 3-D cell-matrix complex. Cells seeded on PET matrices with a lower porosity (similar to 87%) had higher gene expression activities than cells in the matrices with a higher porosity (similar to 90%). Also, Higher gene expression levels of beta -gal were obtained for the more uniformly seeded matrices that were seeded with a depth-filtration method. The results from this study demonstrate the potential utility of cells seeded onto 3-D fibrous matrices as cell-based gene delivery vehicle for in vitro study of gene expression or in vivo gene therapy.
Keywords
*Elastomers, *Polyesters, *Tissue Engineering/instrumentation/methods, Animals, Cell Proliferation, Cell Survival, Cells, Cultured, Chondrocytes/*metabolism, Collagen/metabolism, Extracellular Matrix/*metabolism, Rabbits, RNA, Messenger/*biosynthesis, Stress, Mechanical
*Elastomers, *Polyesters, *Tissue Engineering/instrumentation/methods, Animals, Cell Proliferation, Cell Survival, Cells, Cultured, Chondrocytes/*metabolism, Collagen/metabolism, Extracellular Matrix/*metabolism, Rabbits, RNA, Messenger/*biosynthesis, Stress, Mechanical
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CellLine: NIH 3T3
Morphology: Fibroblast
Origin: Embryo
Species: Mouse
Scaffold Form: fibers/meshMorphology: Fibroblast
Origin: Embryo
Species: Mouse
Scaffold Material: PET/ poly(ethylene terephthalate)
Scaffold Origin: synthetic
Scaffold Permanance: Degradable