Combinations of Osmolytes, Including Monosaccharides, Disaccharides, and Sugar Alcohols Act in Concert During Cryopreservation to Improve Mesenchymal Stromal Cell Survival

Abstract

There is demand for non-dimethyl sulfoxide (DMSO) cryoprotective agents that maintain cell viability without causing poor postthaw function or systemic toxicity. The focus of this investigation involves expanding our understanding of multicomponent osmolyte solutions and their ability to preserve cell viability during freezing. Controlled cooling rate freezing, Raman microscopy, and differential scanning calorimetry (DSC) were utilized to evaluate the differences in recovery and ice crystal formation behavior for solutions containing multiple cryoprotectants, including sugars, sugar alcohols, and small molecule additives. Postthaw recovery of mesenchymal stem cells (MSCs) in solutions containing multiple osmolytes have been shown to be comparable or better than that of MSCs frozen in 10% DMSO at 1°C/min when the solution composition is optimized. Maximum postthaw recovery was observed in these multiple osmolyte solutions with incubation times of up to 2 h before freezing. Raman images demonstrate large ice crystal formation in cryopreserved cells incubated for shorter periods of time (*30 min), suggesting that longer permeation times are needed for these solutions. Recovery was dependent upon the concentration of each component in solution, and was not strongly correlated with osmolarity. It is noteworthy that the postthaw recovery varied significantly with the composition of solutions containing the same three components and this variation exhibited an inverted U-shape behavior, indicating that there may be a ''sweet spot'' for different combinations of osmolytes. Raman images of freezing behavior in different solution compositions were consistent with the observed postthaw recovery. Phase change behavior (solidification patterns and glass-forming tendency) did not differ for solutions with similar osmolarity, but differences in postthaw recovery suggest that biological, not physical, methods of protection are at play. Lastly, molecular substitution of glucose (a monosaccharide) for sucrose (a disaccharide) resulted in a significant drop in recovery. Taken together, the information from these studies increases our understanding of non-DMSO multicomponent cryoprotective solutions and the manner by which they enhance postthaw recovery.

Preservation of cell-based immunotherapies for clinical trials

“…This restriction complicates the workflow considerably at both the site of manufacture and the site of use. DMSO-free multi-osmolyte solutions have effectively preserved MSCs [7] and T lymphocytes [8] and have provided improved cell stability. Therefore, these CPAs have prolonged the window of manufacturing both prefreeze and post-thaw.…”

Characterizing modes of action and interaction for multicomponent osmolyte solutions on Jurkat cells

“…In addition, cell types may respond differently to the same formulation. For example, SMC showed a moderate post‐thaw recovery to mesenchymal stem cells but low post‐thaw recovery in this study (Pollock, Yu et al, ). Glycerol has long been associated with the stabilization of proteins (Timasheff, ).…”

“…The molecular structure of the sugar influences the stabilizing effects. We have established previously that disaccharides are more effective than monosaccharides (Pollock, Yu et al, 2016) in improving the postthaw recovery of cells. Disaccharides can reduce the free water due to hydration effect for proteins in the cell when compared with monosaccharides (Sei et al, 2002;Uchida et al, 2007), suggesting that disaccharides are more effective in stabilizing cell membrane proteins than monosaccharides.…”

“…Although DMSO has been used for cryoprotection for over half century (Lovelock & Bishop, ), it is toxic upon infusion to patients and can result in side effects, such as nausea, vomiting, cardiovascular complication, or even death (Windrum, Morris, Drake, Niederwieser, & Ruutu, ). Crucially, exposure of cells to DMSO can also result in loss of viability and function (Pollock, Yu et al, ). In general, the exposure time for hematopoietic cells is limited to half hour, which adds to the complexity of workflow related to the preservation of cells using DMSO.…”

“…These compounds are used in nature to stabilize biological systems subjected to environmental extremes (P. Yancey, Clark, Hand, Bowlus, & Somero, 1982). In previous studies, we demonstrated that various osmolytes can act in concert to improve cell viability during cryopreservation (Pi, Yu, Petersen, & Hubel, 2018;Pollock, Yu et al, 2016;Pollock et al, 2017;Pollock, Budenske, McKenna, Dosa, & Hubel, 2016;Yu, Yap, Pollock, & Hubel, 2017). We have shown that certain combinations of sugars, sugar alcohols and amino acids not only maintained post-thaw recovery but also resulted in cells that had improved function compared with those that were cryopreserved with DMSO.…”