title: 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.
authors.affiliation: 1 Department of Biomedical Engineering, University of Minnesota , Minneapolis, Minnesota.
authors.forename: Kathryn
authors.initials: K
authors.lastname: Pollock
authors.affiliation: 2 Department of Mechanical Engineering, University of Minnesota , Minneapolis, Minnesota.
authors.forename: Guanglin
authors.initials: G
authors.lastname: Yu
authors.affiliation: 3 Department of Ophthalmology, University of Wisconsin , Madison, Wisconsin.
authors.forename: Ralph
authors.initials: R
authors.lastname: Moller-Trane
authors.affiliation: 1 Department of Biomedical Engineering, University of Minnesota , Minneapolis, Minnesota.
authors.forename: Marissa
authors.initials: M
authors.lastname: Koran
authors.affiliation: 4 Institute for Therapeutics Discovery and Development, University of Minnesota , Minneapolis, Minnesota.
authors.forename: Peter I
authors.initials: PI
authors.lastname: Dosa
authors.affiliation: 5 Department of Laboratory Medicine and Pathology, University of Minnesota , Minneapolis, Minnesota.
authors.forename: David H
authors.initials: DH
authors.lastname: McKenna
authors.affiliation: 2 Department of Mechanical Engineering, University of Minnesota , Minneapolis, Minnesota.
authors.forename: Allison
authors.initials: A
authors.lastname: Hubel
mesh_terms: D002470:Cell Survival; D002478:Cells, Cultured; D015925:Cryopreservation / Q000379:methods*; D003451:Cryoprotective Agents / Q000494:pharmacology*; D004187:Disaccharides / Q000494:pharmacology*; D006801:Humans; D059630:Mesenchymal Stem Cells / Q000166:cytology* / Q000187:drug effects; D009005:Monosaccharides / Q000494:pharmacology*; D009995:Osmosis; D013402:Sugar Alcohols / Q000494:pharmacology*
chemical_list: D003451:Cryoprotective Agents; D004187:Disaccharides; D009005:Monosaccharides; D013402:Sugar Alcohols
keywords: cryopreservation; mesenchymal stromal cell; osmolyte