NMR Assessment of Me 2SO in Decellularized Cryopreserved Aortic Valve Conduits

Abstract

Decellularized cryopreserved allograft vascular tissue may provide a nonimmunogenic scaffold that is suitable for repopulation by cells from a variety of sources, conferring the potential for growth and repair. Although dimethyl sulfoxide (Me(2)SO) is generally regarded as a safe cryoprotectant, even low levels may alter function of repopulating cells. We investigated the residual concentration of Me(2)SO in the aqueous compartment of cryopreserved ovine aortic valve conduits following decellularization. Aortic valve conduits from Suffolk sheep were cryopreserved in 1.1 M (7.5% vol/vol) Me(2)SO according to the protocol of our local tissue bank. Three aortic valve conduits were decellularized in a series of hypotonic and hypertonic Tris buffers. Tissue samples were taken at regular time intervals throughout the decellularization process and equilibrated in double distilled, deionized H(2)O for 28 days. Quantitative proton nuclear magnetic resonance spectroscopy was used to determine the residual Me(2)SO concentration in the equilibration solutions from which Me(2)SO tissue concentrations were calculated. After thawing, the mean Me(2)SO concentration in the valve conduit was 0.302 +/- 0.081 M. The decellularization process resulted in a stepwise reduction in the Me(2)SO concentration to less than 8.56 x 10(-5) +/- 9 x 10(-5) M (P = 0.02). The diffusion coefficient was 2.5 x 10(-6) cm(2)/s. Our study demonstrates that Me(2)SO is effectively washed out of the aortic valve conduit during decellularization, resulting in a final concentration that is several orders of magnitude less than Me(2)SO concentrations reported to alter cell function.