Effectively improved of tissue banking methods for natural and engineered tissues, complex allotransplants and organs are desperately needed for transplantation. Banking of living cellular tissues using current tissue banking practices employing conventional cryopreservation by freezing isn't feasible thanks to the well documented damage caused by ice formation. An alternative ice-free cryopreservation approach is vitrification. The formation of ice can be prevented by the presence of high concentrations of cryoprotectants with preservation of extracellular matrix components and optional preservation of cells. Ice-free vitrification works for a spread of natural and engineered tissues, employing a formulation consisting of DMSO, formamide and propanediol, referred to as VS55, but are unsuccessful at sample volumes over a few mLs. The major constraints for scale-up of cryopreservation by ice-free vitrification have been avoidance of ice nucleation during warming and mechanical forces generated by glasses at low temperatures. In this presentation I will focus on strategies for avoidance of ice nucleation. Our first successful strategy for giant tissue samples was an 83% formulation based upon an equivalent cryoprotectants, referred to as VS83. This formulation can beused to retain viable chondrocytes in large osteochondral grafts or for non-viable cardiovascular grafts with retention of extracellular matrix integrity, depending upon the way in which the formulation is added and removed before and after vitrification. Non-viable cardiovascular grafts with intact matrix have been a major research focus for the last 10 years and both in vitro and in vivo results demonstrated significantly reduced immunogenicity in heart valves (Figure), including reduced memory T-cell proliferation and most recently modulation of TGF-β1 from latent to active form among other statistically significant effects. We have recently been successful in scaling up the viable preservation of large tissue samples using either nano warming, inductive heating of iron nanoparticles, or convection warming using improved ice-free vitrification formulations. Cryopreservation is that the use of very coldness to preserve living cells and tissues during a quiescent status for an extended period, without losing their viability, activity, and performance.
