title: Freeze-thaw increases adeno-associated virus transduction of cells.
abstract: A combination of gene and cell-based therapies may provide significant advantages over existing treatments in terms of their effectiveness. However, long-term efficient gene delivery has been difficult to achieve in many cell types, including endothelial cells. We developed a freeze-thaw technique which significantly increases the transduction efficiency of recombinant adeno-associated virus vectors in human aortic endothelial cells (23-fold) and in human renal proximal tubular epithelial cells (128-fold) in comparison to current methods for transduction. Freeze-thaw resulted in a transient but significant increase in cell surface area by 1,174 +/- 69.8 microM2 per cell. Reduction of cryogenic medium volume and repeated freeze-thaw further increased transduction efficiency by 2.8- and 2.4-fold, respectively. Trypsinization, dimethylsulfoxide, and cold temperatures, which are also involved in cell preservation, had no significant impact on transduction efficiency. Increased transduction was also observed in mesenchymal stem cells (42-fold) by the freeze-thaw method. The potential mechanism of this novel technique likely involves an increase in the net permeable area of biological membranes caused by water crystallization. These findings provide a new approach for gene delivery in various cell types, particularly in those resistant to transduction by conventional methods.
authors.affiliation: Department of Medicine, Nephrology Research and Training Center, Univ. of Alabama at Birmingham, 1530 3rd Ave. S., Birmingham, AL 35294, USA. chens@uab.edu
authors.forename: Sifeng
authors.initials: S
authors.lastname: Chen
authors.forename: Clive
authors.initials: C
authors.lastname: Wasserfall
authors.forename: Matthias H
authors.initials: MH
authors.lastname: Kapturczak
authors.forename: Mark
authors.initials: M
authors.lastname: Atkinson
authors.forename: Anupam
authors.initials: A
authors.lastname: Agarwal
mesh_terms: D000256:Adenoviridae / Q000235:genetics*; D042783:Endothelial Cells / Q000166:cytology / Q000378:metabolism*; D005615:Freezing; D006801:Humans; D007687:Kidney Tubules, Proximal / Q000166:cytology / Q000378:metabolism*; D059630:Mesenchymal Stem Cells / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism*; D014161:Transduction, Genetic / Q000379:methods*; D014162:Transfection / Q000379:methods
chemical_list: D011994:Recombinant Proteins