Antifreeze proteins from polar fish species are remarkable biomacromolecules which prevent the growth of ice crystals. Ice crystal growth is a major problem in cell/tissue cryopreservation for transplantation, transfusion and basic biomedical research, as well as technological applications such as icing of aircraft wings. This review will introduce the rapidly emerging field of synthetic macromolecular (polymer) mimics of antifreeze proteins. Particular focus is placed on designing polymers which have no structural similarities to antifreeze proteins but reproduce the same macroscopic properties, potentially by different molecular-level mechanisms. The application of these polymers to the cryopreservation of donor cells is also introduced. Water is fundamental to all life on our planet, and despite it having a freezing point of 0˚C, Nature has evolved a series of unique adaptations to enable life to flourish in sub-zero climates, at high altitudes and at the Earth's poles. Such extremophiles include the wood frog (Lithobates sylvaticus) which can freeze solid over winter, tardigrades which can be desiccated and rehydrated and cold tolerant plants 1, 2 . The mechanisms of these cryoprotectants are varied, from enabling freeze-tolerance (being able to be frozen and then thawed) to freeze avoidance (preventing ice forming) and even freeze promotion (as a predatory mechanism) 3-6 .One particular adaptation is the production of macromolecular antifreezes (proteins and polysaccharides) which modulate ice formation and growth, and are found in freeze avoidant organisms. These can be broadly split into the antifreeze proteins (AFPs) and antifreeze glycoproteins (AFGPs). AFGPs are highly conserved, with a relatively simple repeat tripeptide structure and a disaccharide on every third amino acid, but are produced in a range of chain lengths. Conversely, AFPs are far more diverse, with several subdivisions, and can assume different structures; from beta barrels to alpha helices and vary in size (Fig. 1). They all have a few core properties (discussed in detail below) including the ability to inhibit ice recrystallization, shape ice crystals into unusual morphologies and to depress the freezing point in a noncolligative manner. The relative magnitude of each effect varies between individual AF(G)Ps and the exact mechanisms, involving ice-face recognition, are still under investigation. What is clear, is that the ability to tune and modify ice growth and formation has the potential for huge industrial and societal impact. For example, ice adhesion limits the performance of wind farms by up to 50% 7 , is a major problem for aircraft 8 and even impacts our understanding of how biological components affect our climate 9, 10 .Some AFPs have already found application in improving the texture of ice cream products by preventing ice crystal growth, and other food uses are under investigation 4,11 . A key potential field where AF(G)Ps could be applied is in cell and tissue cryopreservation where ice

Tissue engineering, gene therapy, drug screening, and emerging regenerative medicine therapies are fundamentally reliant on high‐quality adherent cell culture, but current methods to cryopreserve cells in this format can give low cell yields and require large volumes of solvent “antifreezes”. Herein, we report polyproline as a minimum (bio)synthetic mimic of antifreeze proteins that is accessible by solution, solid‐phase, and recombinant methods. We demonstrate that polyproline has ice recrystallisation inhibition activity linked to its amphipathic helix and that it enhances the DMSO cryopreservation of adherent cell lines. Polyproline may be a versatile additive in the emerging field of macromolecular cryoprotectants.

The storage and transport of frozen cells underpin the emerging/existing cell-based therapies and are used in every biomedical research lab globally. The current gold-standard cryoprotectant dimethyl sulfoxide (DMSO) does not give quantitative cell recovery in suspension or in two-dimensional (2D) or three-dimensional (3D) cell models, and the solvent and cell debris must be removed prior to application/transfusion. There is a real need to improve this 50-year-old method to underpin emerging regenerative and cell-based therapies. Here, we introduce a potent and synthetically scalable polymeric cryopreservation enhancer which is easily obtained in a single step from a low cost and biocompatible precursor, poly(methyl vinyl ether- alt -maleic anhydride). This poly(ampholyte) enables post-thaw recoveries of up to 88% for a 2D cell monolayer model compared to just 24% using conventional DMSO cryopreservation. The poly(ampholyte) also enables reduction of [DMSO] from 10 wt % to just 2.5 wt % in suspension cryopreservation, which can reduce the negative side effects and speed up post-thaw processing. After thawing, the cells have reduced membrane damage and faster growth rates compared to those without the polymer. The polymer appears to function by a unique extracellular mechanism by stabilization of the cell membrane, rather than by modulation of ice formation and growth. This new macromolecular cryoprotectant will find applications across basic and translational biomedical science and may improve the cold chain for cell-based therapies.