title: Three-dimensional cell-scaffold constructs promote efficient gene transfection: implications for cell-based gene therapy.
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 ED(27) 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-betagal and pEGFP (green fluorescent protein) reporter gene vectors using LipofectAmine 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 ( approximately 87%) had higher gene expression activities than cells in the matrices with a higher porosity ( approximately 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.
authors.affiliation: Department of Obstetrics and Gynecology, College of Medicine, The Ohio State University, Columbus, Ohio 43210, USA.
authors.forename: Y
authors.initials: Y
authors.lastname: Xie
authors.forename: S T
authors.initials: ST
authors.lastname: Yang
authors.forename: D A
authors.initials: DA
authors.lastname: Kniss
mesh_terms: D016475:3T3 Cells; D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods; D002460:Cell Line; D018076:DNA, Complementary / Q000235:genetics; D017930:Genes, Reporter; D015316:Genetic Therapy; D049452:Green Fluorescent Proteins; D006801:Humans; D007770:L-Lactate Dehydrogenase / Q000032:analysis; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D018613:Microscopy, Confocal; D011093:Polyethylene Terephthalates / Q000737:chemistry; D013997:Time Factors; D023822:Tissue Engineering / Q000379:methods*; D014162:Transfection / Q000379:methods*; D014327:Trophoblasts / Q000166:cytology / Q000378:metabolism; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
chemical_list: D018076:DNA, Complementary; D008164:Luminescent Proteins; D011093:Polyethylene Terephthalates; D049452:Green Fluorescent Proteins; D007770:L-Lactate Dehydrogenase; D001616:beta-Galactosidase