The Impact of Salts on the Ice Recrystallization Inhibition Activity of Antifreeze (Glyco)Proteins.

Antifreeze (glyco)proteins (AF(G)Ps) have received increasing attention as potential cryopreservation agents since their discovery in the 1970s. While cryopreservation strategies for specific cells (such as red blood cells) are successful and widely implemented, preservation of other cell types, tissues and whole organs remains challenging. This is due to the multifactorial nature of the freeze-thaw damage, the complexity of preserving biological matter and the (country-to-country) variability of the employed procedures and regulations. AF(G)Ps are well-known for their ability to modulate ice crystal growth morphology and ice recrystallization inhibition (IRI), both of which are considered key contributors to freeze-thaw damage. To date, however, the impact of AF(G)Ps on cell survival remains at best partially understood as conflicting results on the benefits or disadvantages of including AF(G)P in cryopreservation strategies remain unelucidated. We hypothesize that variability in the additives in the cryopreservation media contributes to the observed discrepancies. To critically examine this idea, we monitored the inhibition of ice recrystallization by AF(G)P in the presence of various salts using a quantitative analysis of optical microscopy images via the Lifshitz-Slyozov-Wagner (LSW) theory for Oswald ripening. We found that the addition of salts, which are used in culture and cryopreservation media, enhances the IRI activity of AF(G)Ps, and that the magnitude of the enhancement was in line with the Hofmeister series. The size of ice crystals grown in AFGP1-5 and type III AFP samples containing chloride, phosphate and citrate ions were statistically smaller after 90 min of incubation than crystals grown in the absence of these salts. The ice recrystallization rates (kd) of AFGP1-5 and type III AFP samples prepared at a fixed overall ionic strength of 100 mM progressively decreased following the Hofmeister series for anions. Our results demonstrate that the performance of AF(G)Ps is significantly influenced by additives present in common cryopreservation media. It is thus important to conduct excipient compatibility experiments to identify potential incompatibilities between additives and AF(G)Ps in cryopreservation formulations.

Polyproline as a Minimal Antifreeze Protein Mimic That Enhances the Cryopreservation of Cell Monolayers.

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.

Arginine, a key residue for the enhancing ability of an antifreeze protein of the beetle Dendroides canadensis.

Antifreeze proteins (AFPs) can produce a difference between the nonequilibrium freezing point and the melting point, termed thermal hysteresis (TH). The TH activity of an antifreeze protein (AFP) depends on the specific AFP and its concentration as well as the presence of cosolutes including low molecular mass solutes and/or proteins. We recently identified series of carboxylates and polyols as efficient enhancers for an AFP from the beetle Dendroides canadensis. In this study, we chemically modified DAFP-1 using the arginine-specific reagent 1,2-cyclohexanedione. We demonstrated that 1,2-cyclohexanedione specifically modifies one arginine residue and the modified DAFP-1 loses its enhancing ability completely or partially in the presence of previously identified enhancers. The stronger the enhancement ability of the enhancer on the native DAFP-1, the stronger the enhancement effect of the enhancer on the modified DAFP-1. The weaker enhancers (e.g., glycerol) completely lose their enhancement effect on the modified DAFP-1 due to their inability to compete with 1,2-cyclohexanedione for the arginine residue. Regeneration of the arginine residue using hydroxylamine fully restored the enhancing ability of DAFP-1. These studies indicated that an arginine residue is critical for the enhancing ability of DAFP-1 and the guanidinium group of the arginine residue is important for its interaction with the enhancers, where the general mechanism of arginine-ligand interaction is borne. This work may initiate a complete mechanistic study of the enhancement effect in AFPs.