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

Romà Surís-Valls; Ilja K Voets

doi:10.3390/biom9080347

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

Biomolecules. 2019 Aug 6;9(8):347. doi: 10.3390/biom9080347.

Authors

Romà Surís-Valls¹, Ilja K Voets²

Affiliations

¹ Laboratory of Self-Organizing Soft Matter, Laboratory of Macro-Organic Chemistry, Department of Chemical Engineering and Chemistry & Institute for Complex Molecular Systems, Eindhoven University of Technology, Post Office Box 513, 5600 MD Eindhoven, The Netherlands.
² Laboratory of Self-Organizing Soft Matter, Laboratory of Macro-Organic Chemistry, Department of Chemical Engineering and Chemistry & Institute for Complex Molecular Systems, Eindhoven University of Technology, Post Office Box 513, 5600 MD Eindhoven, The Netherlands. i.voets@tue.nl.

Abstract

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 AFGP_1-5 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 (k_d) of AFGP_1-5 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.

Keywords: antifreeze proteins; hofmeister series; ice recrystallization inhibition.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Antifreeze Proteins / antagonists & inhibitors*
Antifreeze Proteins / chemistry
Cryopreservation*
Cryoprotective Agents / chemistry
Cryoprotective Agents / pharmacology*
Crystallization
Ice*
Salts / chemistry
Salts / pharmacology

Substances

Antifreeze Proteins
Cryoprotective Agents
Ice
Salts