010 The control of ice nucleation and growth in tissues and organs

Abstract

Vitrification is a promising general approach to cryopreservation of clinically relevant tissues and organs that are sensitive to ice formation. However, the vitrification of larger samples requires cryoprotectant concentrations sufficiently high to avoid ice formation at relatively low cooling and warming rates. In an effort to avoid the toxicity that usually accompanies higher concentrations, certain extracellular additives have been identified that can improve vitrification tendency while minimizing toxicity, not just in solution but also in whole tissues and organs. Such additives act either through interaction with water in bulk solution (a class we call specialty cryoprotectants) or through apparently specific interactions with ice-like surfaces (a class we call ice blockers). Ice blockers act either through binding to ice nucleators (anti-nucleators) or by binding directly to ice (ice growth inhibitors, whose apparent generalized nucleation inhibition effect is a side effect of ice crystal binding). Unlike specialty cryoprotectants, ice blockers, presumably due to their specificity, tend to be active even at low concentrations (e.g., 0.1% w/v or less), and this property is strong evidence that a compound is an ice blocker rather than a specialty cryoprotectant. Antifreeze proteins are the prototypical and most well-known ice-active ice blockers, but their high cost and other potential disadvantages have stimulated interest in smaller non-protein alternatives. Certain flavonol glycosides and the polymer polyglycerol (PGL) are nucleation inhibitors that are active at low concentrations. More recently polyampholyte polymers with a 65% carboxylate mole fraction and polyglycidol block copolymers have been shown to have ice blocking activity. Polyvinyl alcohol (PVA), originally thought to be only a nucleation inhibitor, has since been shown in the literature to exhibit recrystallization inhibition, thermal hysteresis, and altered ice crystal morphology in its thermal hysteresis zone, all characteristics previously only observed for antifreeze proteins. PVA also raises the homogeneous nucleation temperature of water in emulsions, a behavior consistent with a structural match to ice at nanometer scales. Molecular dynamics simulations retrospectively showed that a minimum energy conformation of PVA can bind to the basal plane of ice, with hydrophobic methylene groups positioned to intercalate between water molecules in the basal plane. Zirconium acetate, which also alters ice crystal morphology and inhibits recrystallization, was also proposed to be active through bonding to the ice lattice. The diequatorial conformation of cis-1,3-cyclohexandiol (CHD) has also generated interest from the prospective observation that its hydroxyls are favorably positioned for binding to water molecules in the basal plane. One half molar (7.6% w/v) CHD was shown to almost double the thermal hysteresis activity of an antifreeze glycoprotein compared to no effect using 1,4-cyclohexanediol as a control, and 6% w/v CHD added to a vitrification solution also greatly slowed the growth of ice compared to 6% sucrose. However, some conventional solutes such as glycerol and propylene glycol can have similar effects, and the need for multi-percent concentrations is typical for a specialty cryoprotectant. In conclusion, ice blockers and specialty cryoprotectants are becoming increasingly indispensable and increasingly interesting additives for large-scale cryopreservation by vitrification. Source of funding: Supported by 21st Century Medicine, Inc. Conflict of interest: 21st Century Medicine is a supplier of PGL and PVA ice blockers. gfahy@21cm.com