Submitted on November 21, 2005
Accepted on February 22, 2006
Freeze-thaw increases adeno-associated virus transduction of cells
Sifeng Chen1*, Clive Wasserfall2, Matthias H Kapturczak1, Mark Atkinson2, and Anupam Agarwal1
1 Medicine, University of Alabama at Birmingham, Birmingham, AL, USA
2 Pathology, University of Florida, Gainesville, FL, USA
* To whom correspondence should be addressed. E-mail: chens{at}uab.edu.
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) comparison to current methods for transduction. Freeze-thaw resulted in a transient but significant increase in cell surface area by 1174 ± 69.8 µM2 per cell. Reducing cryogenic medium volumes and repeated freeze-thaw further increased transduction efficiency by 2.8- and 2.4-fold, respectively. Trypsinization, dimethylsulfoxide and cold temperature, 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.
