Three-dimensional cell-scaffold constructs promote efficient gene transfection: Implications for cell-based gene therapy

To date, introduction of gene-modified cells in vivo is still a critical li mitation for cell-based gene therapy. In this study, based on tissue engine ering techniques, we developed a three-dimensional (3-D) transfection syste m to be cell-based gene delivery vehicle. Human trophoblast-like ED27 and f ibroblastic NIH3T3 cells were used as model cell lines. Cells were seeded o nto PET fibrous matrices and plated on polyethylene terephathalate (PET) fi lms as 2-D transfection control. The cell-matrices and cell-films were tran sfected with pCMV-beta gal and pEGFP (green fluorescent protein) reporter g ene 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 express ion time as compared to 2-D transfection. There existed an optimal initial cell seeding density for gene transfection of 3-D cell-matrix complex. Cell s seeded on PET matrices with a lower porosity (similar to 87%) had higher gene expression activities than cells in the matrices with a higher porosit y (similar to 90%). Also, Higher gene expression levels of beta -gal were o btained for the more uniformly seeded matrices that were seeded with a dept h-filtration method. The results from this study demonstrate the potential utility of cells seeded onto 3-D fibrous matrices as cell-based gene delive ry vehicle for in vitro study of gene expression or in vivo gene therapy.