title: Efficient Gene Transduction of Dispersed Islet Cells in Culture Using Fiber-Modified Adenoviral Vectors.
abstract: To establish novel islet-based therapies, our group has recently developed technologies for creating functional neo-islet tissues in the subcutaneous space by transplanting monolithic sheets of dispersed islet cells (islet cell sheets). Improving cellular function and viability are the next important challenges for enhancing the therapeutic effects. This article describes the adenoviral vector-mediated gene transduction of dispersed islet cells under culture conditions. Purified pancreatic islets were obtained from Lewis rats and dissociated into single islet cells. Cells were plated onto laminin-5-coated temperature-responsive polymer poly(N-isopropylacrylamide)-immobilized plastic dishes. At 0 h, islet cells were infected for 1 h with either conventional type 5 adenoviral vector (Ad-CA-GFP) or fiber-modified adenoviral vector (AdK7-CA-GFP) harboring a polylysine (K7) peptide in the C terminus of the fiber knob. We investigated gene transduction efficiency at 48 h after infection and found that AdK7-CA-GFP yielded higher transduction efficiencies than Ad-CA-GFP at a multiplicity of infection (MOI) of 5 and 10. For AdK7-CA-GFP at MOI = 10, 84.4 ± 1.5% of islet cells were found to be genetically transduced without marked vector infection-related cellular damage as determined by viable cell number and lactate dehydrogenase (LDH) release assay. After AdK7-CA-GFP infection at MOI = 10, cells remained attached and expanded to nearly full confluency, showing that this adenoviral infection protocol is a feasible approach for creating islet cell sheets. We have shown that dispersed and cultured islet cells can be genetically modified efficiently using fiber-modified adenoviral vectors. Therefore, this gene therapy technique could be used for cellular modification or biological assessment of dispersed islet cells.
authors: Department of Regenerative Surgery, Fukushima Medical University , Hikarigaoka, Fukushima , Japan.
authors: Hiroyuki
authors: H
authors: Hanayama
authors: † iPS Cell-based Projects on Cell Transplantation and Cell Dynamics, Graduate School of Pharmaceutical Sciences, Osaka University , Suita, Osaka , Japan.
authors: Kazuo
authors: K
authors: Ohashi
authors: ‡ Institute of Advanced Biomedical Engineering and Science, Tokyo Women's Medical University , Shinjuku, Tokyo , Japan.
authors: Rie
authors: R
authors: Utoh
authors: Department of Regenerative Surgery, Fukushima Medical University , Hikarigaoka, Fukushima , Japan.
authors: Hirofumi
authors: H
authors: Shimizu
authors: Department of Regenerative Surgery, Fukushima Medical University , Hikarigaoka, Fukushima , Japan.
authors: Kazuya
authors: K
authors: Ise
authors: § Laboratory of Biochemistry and Molecular Biology, Graduate School of Pharmaceutical Sciences, Osaka University , Suita, Osaka , Japan.
authors: Fuminori
authors: F
authors: Sakurai
authors: § Laboratory of Biochemistry and Molecular Biology, Graduate School of Pharmaceutical Sciences, Osaka University , Suita, Osaka , Japan.
authors: Hiroyuki
authors: H
authors: Mizuguchi
authors: † iPS Cell-based Projects on Cell Transplantation and Cell Dynamics, Graduate School of Pharmaceutical Sciences, Osaka University , Suita, Osaka , Japan.
authors: Hiroyuki
authors: H
authors: Tsuchiya
authors: ‡ Institute of Advanced Biomedical Engineering and Science, Tokyo Women's Medical University , Shinjuku, Tokyo , Japan.
authors: Teruo
authors: T
authors: Okano
authors: Department of Regenerative Surgery, Fukushima Medical University , Hikarigaoka, Fukushima , Japan.
authors: Mitsukazu
authors: M
authors: Gotoh
keywords: Dispersed islet cell; Fiber-modified adenoviral vector; Gene therapy; Islet transplantation; Tissue engineering