| Summary: | Cryopreservation is a vitally important field of study, and although significant advances have been made in the last 60 years, many challenges still exist. The exact mechanisms by which injury occurs are still debated, and therefore the design of cryoprotectants is uniquely challenging. A key event during cryopreservation is the recrystallization of ice, which results in damage to cells. Antifreeze glycoproteins (AFGPs) are a subclass of biological antifreezes found in many species of Arctic and Antarctic teleost fish, and these possess potent recrystallization inhibition (RI) activity. Consequently, our lab has explored the rational design of chemically and biologically stable AFGP analogues for use in cryopreservation. In this study, we have prepared C-linked AFGP analogues and their derivatives to probe the mechanism of RI, and explored the relationship between RI activity and cryopreservation. Recently, our lab prepared a series of C-linked AFGP analogues substituted with different monosaccharides in order to explore the connection between the stereochemistry of the carbohydrate moiety and recrystallization activity. A subsequent study of a series of commercially available O-linked mono- and disaccharides confirmed that the hydration of the carbohydrate, which is determined by its stereochemistry, is correlated to its RI activity. The hydration of a compound is further related to its hydrophobicity. Consequently, we designed a series of analogues to investigate the importance of hydrophobicity and hydration of C-linked AFGP analogues and their components to RI activity. AFGP analogues substituted with a carbohydrate moiety containing a double bond, AFGP analogue building blocks substituted with both saturated and unsaturated carbohydrate moieties, and a series of C-linked carbohydrate derivatives bearing a variety of hydrophobic groups were prepared and tested for RI activity. The data obtained reinforced the importance of stereochemistry for RI activity. The double bond would have disturbed the stereochemistry of the hydroxyl groups compared to the saturated carbohydrate, and this resulted in a decrease in RI activity. Further, it was found that increasing the hydrophobicity of C-linked carbohydrate derivatives does not necessarily improve RI activity. Lastly, the AFGP analogues had much greater RI activity than their individual components, and the components cannot be used to predict the RI activity of the polymer. Our cryopreservation studies were carried out using two cell types, a hepatocyte-like cell line (WRL 68), and hematopoietic stem cells (HSCs) from umbilical cord blood (UCB). Hematopoietic stem cells (HSCs) are immature hematopoietic precursor cells also found in bone marrow and peripheral blood. Transplantation of both HSCs and hepatocytes is indicated for myriad conditions, and a reliable supply of cells is paramount. There is a need for improved cryopreservation conditions for both cell types to achieve this goal. Based on the extensive work done by our lab on the effect of hydration of carbohydrates on RI, we investigated the cryopreservation potential of several carbohydrates, representing a spectrum of RI activity. It was found that that low concentrations of carbohydrates can be used for successful cryopreservation of WRL 68 cells, and further, the addition of low concentration of carbohydrates can allow the concentration of DMSO in the cryopreservation medium to be reduced without adversely affecting the cryopreservation outcome. For UCB cells, 5% DMSO can be completely replaced with galactose, without a decrease in cell viability. For both cell types, in the presence of DMSO, the type of carbohydrate was more important than its concentration for a successful cryopreservation outcome. Further, carbohydrates are most effective when employed as extracellular cryoprotectants. Significantly, when used as the sole cryoprotectant, we determined that carbohydrate cryopreservation activity correlated with RI activity. Carbohydrates with good RI activity may therefore be able to effectively manage the ice formed during cryopreservation.
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