Bruno M. Guerreiro, PhD Student of the Biochemical Engineering Lab at UCIBIO-NOVA School of Science and Technology, pioneered the research and understanding of biotechnologically produced carbohydrate polymers in isochoric cryopreservation. He found out that the fucose-rich FucoPol can control the nucleation stage of ice growth, which is important for designing biopreservation protocols for practical use. The results of this research work were published in the journal ACS Biomaterials Science & Engineering: “Enhanced Control over Ice Nucleation Stochasticity Using a Carbohydrate Polymer Cryoprotectant”. The insights obtained by Bruno’s work granted him the 2022 John K. Critser Award from The Society for Cryobiology and were recently featured in Forbes as exciting advances in cryobiology, its future potential, and how it could save lives.
Bruno Guerreiro’s PhD work focuses on understanding how and why a polymer can be cryoprotective. In cryopreservation, cells, tissues, organs, and whole organisms alike, are always frozen exposed to small molecules capable of inhibiting the lethal ice growth. However, all cryoprotectants known to date, including the commonly used gold standards glycerol and DMSO, are only effective at concentrations that are cytotoxic”. “I had consistently shown that polysaccharides produced by bacteria, algae and other sources do have cryoprotective function, mostly attributed to their high viscosity and negative charge, which can disrupt the organization of water molecules into stable ice crystals, much like how a natural antifreeze protein works. Bio-based polymers are exciting alternatives for their added-value production and inherent biocompatibility, eliminating the toxicity issue”, explains Bruno Guerreiro.
With financial support of the Fulbright Portugal Program, the Luso-American Development Foundation (FLAD), and the Company of Biologists, Bruno Guerreiro collaborated with the University of California, Berkeley to study the effects of the cryoprotective polysaccharide FucoPol in the nucleation stage of isochoric cryopreservation. Water has a negative thermal expansion coefficient, which means that, when frozen, ice occupies a bigger volume than the liquid phase. In an isochoric chamber filled with water, ice expansion cannot proceed to the full extent because (unlike conventional freezers) the recipient is non-deformable, so the system will equilibrate as a two-phase system of partially frozen water. Most focus in the field is on avoiding crystallization, but a fully ice-free system is thermodynamically impossible. But in an isochoric system, biological matter can be spatially separated from crystals without breaking any laws of thermodynamics. Bruno Guerreiro adds “for instance, by placing a crystal seed in bottom of the chamber, partial freezing will occur and halt at thermodynamic equilibrium without ever touching a precious heart suspended on top. But although the extension and location of ice formation can be fine-tuned, nucleation is still the weakness of isochoric processes. Nucleation is a stochastic event, which means that molecular aggregation will occur in a known temperature range, but the single temperature at which freezing will boom cannot be predicted. So, when liquid water is being cooled, what determines when and how much the system will freeze is the nucleation temperature.”. Bruno Guerreiro knew that unlike most cryoprotectants that delay crystal formation in time by creating supercooled water, FucoPol actually decreases the size of ice crystals formed by 10x by accelerating crystallization at higher freezing temperatures. But now, he has discovered that FucoPol can also influence nucleation. Pure water will nucleate within a 6ºC interval (between -9 and -15 ° C), but with FucoPol, this interval narrows to 2 ° C, but the mean nucleation temperature (-11 ° C) is unchanged. This major reduction in the nucleation range makes FucoPol the first stochasticity-regulating polymer to be found. Additionally, FucoPol drastically stabilizes the supercooled state of water, exponentially increasing the induction time necessary for water to nucleate and kickstart ice growth.
This is an emergent discovery in the design of biopreservation protocols because it finally gives power to the cryobiologist. Traditionally, one would immerse the biologics in a preservation solution, freeze to -80ºC and hope for the best. Success would be statistically measured, but real-world problems in the field require success to be a palpable individual outcome: either the organ lives or is lost forever. Now, a cryobiologist trying to isochorically preserve a critical transplant organ at, for example, -14 ° C in pure water (or in any other cryoprotectant), would be at a very high risk of lethal, low-probability rogue nucleation events; but would be quite safe using a FucoPol-containing formula, because the nucleation interval ranges from -9.5 to -11.5 ° C. Nucleation, once thought to be an inevitable phenomenon that left us at its mercy, can now be controlled probabilistically. With pure water alone, an organ placed at -8ºC will probably nucleate in 1 hour, but with 0.25% FucoPol, it will take two weeks until ice forms. This trend is exponential, because the use of 0.5% FucoPol means that an organ can remain cryopreserved in an ice-free environment for a full year. This means that, finally, we are no longer subject to the time it takes for a donor organ to reach its receptor patient. We can execute planned preservation procedures without the organ becoming inviable. Thus, FucoPol, and its cryoprotective properties show exciting promise in the development of biocompatible, safe, and effective biopreservation formula and protocol design.
Enhanced Control over Ice Nucleation Stochasticity Using a Carbohydrate Polymer Cryoprotectant
Bruno M. Guerreiro, Anthony N. Consiglio, Boris Rubinsky, Matthew J. Powell-Palm*, and Filomena Freitas*
ACS Biomater. Sci. Eng. 2022, 8, 5, 1852–1859
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