Fight Aging!

Ice crystal formation is one of the big challenges in low-temperature tissue preservation. Ideally one wants vitrification rather than freezing. The former is the formation of a glass-like state in which even very fine-scale structure is preserved, such as axonal connections between neurons. The latter produces ice crystal formation that is disruptive to small-scale structures such as cells and their organelles. Existing cryoprotectants are good at their task of preventing ice crystal formation if they can be perfused through the whole tissue, which is unfortunately by no means a given in large tissue sections using existing techniques, at least if the tissue is to remain viable as a structure. Also unfortunately, these cryoprotectants are largely quite toxic.

These and related considerations are why there is a drive to produce better cryoprotectants. Mining the natural world for proteins that prevent ice crystal formation may open the door to molecules that can better spread through living tissues prior to harvest and cryopreservation, and some of these proteins are already better in some respects than the artificial cryoprotectants used in research. This isn't just a matter of better logistics for research samples. It isn't just a matter of finding ways to make the organ transplant industry more efficient, and allow donor organs to be stored indefinitely. It is also important to the field of cryonics, the low-temperature preservation of the brain and body at death, in order to offer those individuals a chance of restoration in a more technologically capable future.

At present, perfusing existing cryoprotectants into an entire body effectively immediately following clinical death is challenging. Parts of the brain and body may receive too little cryoprotectant and be vulnerable to ice-crystal formation. If a non-toxic cryoprotectant protein could be delivered systemically over a period of time prior to clinical death, this delivery issue could be solved: the patient would just have to be promptly cooled. This point of starting preparation well prior to clinical death is a strong theme across the board in cryonics. Time matters greatly when it comes to prevention of tissue loss in the brain after clinical death, and the worst thing that can happen is an unexpected, unprepared need for cryopreservation. Delay and cost are the almost least worst of the poor outcomes that can result.

Extended Temperature Range of the Ice-Binding Protein Activity

Cryopreservation is currently the main method for the long-term storage of cells and tissues. At extremely low temperatures, the diffusion is slow, and molecules do not have enough energy to pass energy barriers for chemical reactions. Therefore, biological activity practically ceases, and the cells and tissues can be preserved. However, ice growth during the cooling and warming stages poses a significant challenge. Intracellular freezing is usually considered to be lethal. Extracellular ice growth leads to water depletion from the solutions, resulting in an elevated solute concentration and diffusion of water out of the cells. This leads to osmotic stress due to heightened intracellular solute concentration, membrane injuries, and physical stress on shrinking cells. Ice recrystallization (IR), the process of enlargement of ice crystals at the expense of smaller crystals, is considered damaging and occurs during the freezing and thawing. The amount of ice and its growth pattern are contingent on the solutes and on the temperature profile through freezing, storage, and thawing.

The primary approach for mitigating ice growth damage in cryopreservation is through vitrification. Vitrification is the conversion of a liquid to an amorphous solid glass without undergoing crystallization. This process occurs through rapid cooling, effectively bypassing the ice growth and nucleation zones between the melting temperature (Tm) and the glass-transition temperature (Tg). The liquid water molecules do not have sufficient time to organize into a crystalline structure and rigidify into a glass state with exceptionally high viscosity. When the target is much larger than a single cell, it is impractical to obtain stable vitrification solely by fast cooling and heating. Vitrification of biological samples involves a combination of rapid cooling and heating rates, in addition to adding cryoprotective agents (CPAs). CPAs depress the melting temperature (Tm) and the homogeneous nucleation temperature (Th) while also elevating the Tg in a concentration-dependent manner. This results in a narrower temperature difference between Tm and Tg, effectively reducing the ice growth and nucleation phases and enabling vitrification at slower cooling rates.

One such approach to mitigate devitrification involves the introduction of various ice-active substances. Ice-binding proteins (IBPs), as suggested by their name, possess an inherent capability to bind to ice crystals and nuclei, aiding organisms in surviving freezing conditions. Through direct interaction with water molecules on the ice surface or at the ice-water interface, IBPs exert significant physical effects on the subsequent growth of the bound ice crystal. IBPs depress the freezing point of an ice crystal in a noncolligative manner by blocking the access of water molecules to the ice surface, resulting in a lower freezing point than the melting point within an IBP solution. This mode of ice growth inhibition markedly differs from the colligative effect of small molecule CPAs used in vitrification. Moreover, IBPs exhibit robust IR inhibition activities.

This study investigates the impact of two distinct IBP types on vitrified DMSO solutions at concentrations relevant to cryopreservation procedures. The IBPs used in our research are antifreeze proteins (AFPs), which are a subset of IBPs that particularly act to depress ice growth and recrystallization. We investigate the impact of two types of antifreeze proteins (AFPs): type III AFP from fish and a hyperactive AFP from an insect, the Tenebrio molitor AFP. We report that these AFPs depress devitrification at -80 °C. Furthermore, in cases where devitrification does occur, AFPs depress ice recrystallization during the warming stage. The data directly demonstrate that AFPs are active at temperatures below the regime of homogeneous nucleation. This research paves the way for exploring AFPs as potential enhancers of cryopreservation techniques, minimizing ice-growth-related damage, and promoting advancements in this vital field.