22. Avoiding the problems of ice in tissues via vitrification

Cryopreservation involving freezing in the presence of protective agents such as dimethyl sulfoxide is a well-established method for storing cells and small tissues. While successful techniques for cryopreservation have been developed over the past seven decades, they are generally related to small specimens, in the scale-range of cell suspensions and clusters to small-organized tissues (μm to mm range), with blood cells, stem cells, embryos and pancreatic islets as examples. Cryopreservation of larger-size specimens (cm and above) has been accomplished only in cases where the mechanical functionality has a higher priority need than the recovery of biological functionality, as with heart valves. There is evidence that ice formation within the extracellular matrix of multicellular tissues is the principal event that limits the survival of cryopreserved tissues using conventional freezing techniques. This mode of injury can be circumvented by using an ice-free cryopreservation approach, known as vitreous cryopreservation. Vitrification, the amorphous solidification of a supercooled liquid, can be achieved by adjusting the solute composition and thermal processing rates such that nucleation and growth of ice crystals are essentially prevented. Avoidance of ice by vitrification can be generally achieved with one of two approaches or a combination of both. The first approach employs cooling highly concentrated solutions (typically >50% w/w) that become sufficiently viscous at low temperatures to suppress crystallization rates. Typically, a vitrified material is considered solid when the viscosity reaches 1015 poise. Vitrification can also be achieved by selecting sufficiently high cooling rates to prevent ice crystallization in relatively dilute solutions (<50% w/w). This second approach generally produces a metastable state that is at risk of devitrification and/or recrystallization during warming. Ice formation during warming is just as potentially injurious as during cooling. Applications of the vitrification approach to the cryopreservation of viable tissues such as blood vessels and cartilage has been demonstrated to result in a dramatic improvement in post-rewarm functionality from a meagre 20% after classical freezing to a respectable >80% after ice-free cryopreservation. These achievements provide confidence and enthusiasm that this approach holds great promise for future cryobanking of complex bulky tissues and organs, with an isolated case of kidney cryopreservation having been reported. Nevertheless there are well recognized challenges that remain to be investigated and resolved. While vitrification is a well-understood phenomenon, its application to biological systems comes with potentially harmful effects of toxicity of the cryoprotectants and structural damage due to thermo-mechanical stresses. In fact, these effects represent competing needs important for selecting cryoprotectants and their concentrations, and represent a significant barrier to the development of cryopreservation technology. New approaches to alleviate this couple molecules that modulate ice nucleation and growth with the cryoprotectant cocktail. Both natural and synthetic compounds are being explored as a new class of additives that are anticipated to be essential for ice-free cryopreservation of bulky tissues and organs. In addition, new innovative technologies are conceived to address the challenges associated with the needed for uniform heating of vitrified tissues to avoid the problems of devitrification and recrystallization during warming.