56. Poly-vitrification – A new approach to organ preservation

Abstract

Vitrification has long promised to revolutionize organ cryopreservation and banking, but successful vitrification of complex tissues remains elusive. The two pillars of vitrification – high CPA concentrations coupled with rapid cooling/warming rates – are incompatible for large volume cryopreservation. Challenges include (1) delivery of sufficiently high CPA concentrations homogeneously throughout the entire organ prior to onset of CPA-induced toxicity and (2) uneven cooling and warming rates. To overcome these challenges in whole organ cryopreservation, we propose the concept of a CPA cocktail whose constituents undergo reversible polymerization-like reactions at low temperatures. The cocktail would remain fluid and easily perfusable at high-subzero temperatures, but rapidly increases in viscosity at lower temperatures, above and beyond that seen with individual CPAs. To our knowledge, this approach to organ cryopreservation has not been discussed in the literature. We call this method poly-vitrification, for polymerization-enhanced vitrification. Systems showing polymerization-like effects have been observed in nature, namely with the Goldenrod Gall Fly, which relies on interactions between fructose, glycerol and trehalose. Here, a relatively low concentration of fructose (0.011 M) drives extensive interactions with glycerol and trehalose, forming a partial glassy state at approximately −25 °C, well above the sublimation point of CO2. The benefits of poly-vitrification are two-fold: first, the temperature of the glass transition could perhaps be raised to sub-zero temperatures well above the sublimation point of CO2. This would hypothetically reduce the temperature range for which cooling is required, minimizing toxic exposure to cryoprotectants during cooling and warming. With a higher vitrification temperature, organ transport teams could use dry ice to transport organs to and from areas without LN2 infrastructure. Second, this polymerization-like process would reduce the total concentration of CPAs to perhaps only 1-2 Molar, greatly reducing the effect of CPA toxicity. For poly-vitrification to be successful, extra- and intra-cellular CPAs would have to be equilibrated to ensure vitrification extra- and intra-cellularly occurs at the same time. Methods to load cells with impermeable or slowly permeating molecules, such as fructose, would have to be further developed, perhaps through sugar loaded liposomes, micelles, or an enzymatically triggered CPA-delivery platform built on existing technologies. Alternatively or in conjunction with these methods, synthetic molecules analogous to the natural poly-vitrification systems could be developed. Molecular screening is a widespread approach to developing such synthetic compounds, and warrants further attention in cryobiology. We hypothesize that poly-vitrification could be used to both reduce CPA concentration and toxicity and allow vitrification at much higher temperatures. This will enable reliable and robust organ and tissue preservation to become widespread.