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Ice recrystallization is strongly inhibited when antifreeze proteins bind to multiple ice planes.

ABSTRACT: Ice recrystallization is a phenomenon observed as the increase in ice crystal size within an already frozen material. Antifreeze proteins (AFPs), a class of proteins capable of arresting ice crystal growth, are known to inhibit this phenomenon even at sub milli-molar concentrations. A tremendous range in the possible applications of AFPs is hence expected in both medical and industrial fields, while a key determinant of the ice recrystallization inhibition (IRI) is hardly understood. Here, IRI efficiency and ice plane affinity were examined for the wild-type AFPI-III, a defective AFPIII isoform, and a fungal AFP isoform. To simplify the IRI analysis using the formal representation of Ostwald-ripening (r3?=?r03?+?kt), we monitored specific ice grains exhibiting only uniform growth, for which maximum Feret diameter was measured. The cube of an ice grain's radius (r3) increased proportionately with time (t), and its slope gave the recrystallization rate (k). There was a significant difference in the IRI efficiency between the samples, and the fungal AFP possessing the activity with the smallest amount (0.27??M) exhibited an affinity to multiple ice planes. These results suggest that the IRI efficiency is maximized when AFPs bind to a whole set of ice planes.

SUBMITTER: Rahman AT

PROVIDER: S-EPMC6374469 | biostudies-literature | 2019 Feb

REPOSITORIES: biostudies-literature

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Project description: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.

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Ice recrystallization is strongly inhibited when antifreeze proteins bind to multiple ice planes.

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