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 μM2per 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.