Small-Molecule Ice Recrystallization Inhibitors Improve the Post-Thaw Function of Hematopoietic Stem and Progenitor Cells

The success of hematopoietic stem cell transplantation depends in part on the number and the quality of cells transplanted. Cryoinjuries during freezing and thawing reduce the ability of hematopoietic stem and progenitor cells (HSPCs) to proliferate and differentiate after thawing. Up to 20% of the patients undergoing umbilical cord blood (UCB) transplant experience delayed or failed engraftment, likely because of the inadequate hematopoietic potency of the unit. Therefore, the optimization of cryopreservation protocols, with an emphasis on the preservation of HSPCs, is an important issue. Current protocols typically utilize a 10% dimethyl sulfoxide cryoprotectant solution. This solution ensures 70–80% post-thaw cell viability by diluting intracellular solutes and maintaining the cell volume during cryopreservation. However, this solution fails to fully protect HSPCs, resulting in the loss of potency. Therefore, a new class of cryoprotectants (<i>N</i>-aryl-d-aldonamides) was designed and assessed for the ability to inhibit ice recrystallization and to protect HSPCs against cryoinjury. Several highly active ice recrystallization inhibitors were discovered. When used as additives to the conventional cryoprotectant solution, these nontoxic small molecules improved the preservation of functionally divergent hematopoietic progenitors in the colony-forming unit and long-term culture-initiating cell assays. By contrast, structurally similar compounds that did not inhibit ice recrystallization failed to improve the post-thaw recovery of myeloid progenitors. Together, these results demonstrate that the supplementation of cryopreservation solution with compounds capable of controlling ice recrystallization increases the post-thaw function and potency of HSPCs in UCB. This increase may translate into reduced risk of engraftment failure and allow for greater use of cryopreserved cord blood units

Quantitative Analysis of the Efficacy and Potency of Novel Small Molecule Ice Recrystallization Inhibitors

The effects of ice recrystallization are well-recognized throughout the literature. This phenomenon is the major cause for cellular damage during thawing of cells, ultimately reducing post-thaw viability and function. In this paper, we describe a method for quantifying the inhibitory effect on ice recrystallization of novel small molecules that are cryoprotectants for red blood cells. The method is ideally suited to the splat-cooling assay, where high-ice volume fractions are present. Using our method, we have derived first-order rate constants for the increase in the average crystal size based upon a “binning” approach of ice crystals as a function of size and time. Using this reliable metric, dose–response curves were constructed to obtain IC₅₀ values. Two very effective inhibitors of ice recrystallization, <i>p</i>-methoxyphenyl β-d-glucopyranoside (PMP-Glc) and <i>p</i>-bromophenyl β-d-glucopyranoside (pBrPh-Glc), had IC₅₀ values of 16.3 and 14.8 mM, respectively. Interestingly, the Hill slopes from these dose–response curves were 5.12 ± 0.81 for PMP-Glc and 3.12 ± 0.62 for pBrPh-Glc, suggesting that an element of cooperativity may be involved in the mechanism by which these compounds inhibit ice recrystallization. This is particularly interesting, as unlike antifreeze (glyco)­proteins, these small molecules do not bind to the ice surface

<i>O</i>‑Aryl-Glycoside Ice Recrystallization Inhibitors as Novel Cryoprotectants: A Structure–Function Study

Low-molecular-weight ice recrystallization inhibitors (IRIs) are ideal cryoprotectants that control the growth of ice and mitigate cell damage during freezing. Herein, we describe a detailed study correlating the ice recrystallization inhibition activity and the cryopreservation ability with the structure of <i>O</i>-aryl-glycosides. Many effective IRIs are efficient cryoadditives for the freezing of red blood cells (RBCs). One effective cryoadditive did not inhibit ice recrystallization but instead inhibited ice nucleation, demonstrating the significance of inhibiting both processes and illustrating the importance of this emerging class of cryoprotectants