Effect of trehalose as an additive to dimethyl sulfoxide solutions on ice formation,cellular viability,and metabolism |
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| Institution: | 1. Department of Mechanical Engineering, University of Michigan-Dearborn, 4901 Evergreen Road, Dearborn, MI 48128, United States;2. Department of Biology, University of Louisville, Louisville, KY 40292, United States;1. Department of Mechanical Engineering, University of Michigan-Dearborn, 4901 Evergreen Road, Dearborn, MI 48128, United States;2. Department of Biology, University of Louisville, Louisville, KY 40292, United States;1. State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi 214122, China;2. State Key Laboratory of Dairy Biotechnology, Shanghai Engineering Research Center of Dairy Biotechnology, Dairy Research Institute, Bright Dairy & Food Co., Ltd., Shanghai 200436, China;3. School of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu 214122, China;4. National Engineering Research Center for Functional Food, Jiangnan University, Wuxi, Jiangsu 214122, China;5. Beijing Innovation Centre of Food Nutrition and Human Health, Beijing Technology & Business University, Beijing 100048, China;1. Institute for Multiphase Processes, Leibniz Universität Hannover, Hannover, Germany;2. Service Unit Embryonic Stem Cells, Institute for Transfusion Medicine, Medical School Hannover, Germany;1. Biopharma Technology Limited, Winchester SO23 0LD, United Kingdom;2. Universitat des Saarlandes, Experimental Physics, 66123 Saarbruecken, Germany;3. Universitat Rovira i Virgili (URV), Tarragona 43007, Spain;4. CIRIMAT Institute, Université de Toulouse, CNRS, INPT, UPS, Ensiacet, 4 allée E. Monso, 31030 Toulouse, France;5. Advanced Materials Department, Jozef Stefan Institute, 1000 Ljubljana, Slovenia |
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| Abstract: | Cryopreservation is the only established method for long-term preservation of cells and cellular material. This technique involves preservation of cells and cellular components in the presence of cryoprotective agents (CPAs) at liquid nitrogen temperatures (?196 °C). The organic solvent dimethyl sulfoxide (Me2SO) is one of the most commonly utilized CPAs and has been used with various levels of success depending on the type of cells. In recent years, to improve cryogenic outcomes, the non-reducing disaccharide trehalose has been used as an additive to Me2SO-based freezing solutions. Trehalose is a naturally occurring non-toxic compound found in bacteria, fungi, plants, and invertebrates which has been shown to provide cellular protection during water-limited states. The mechanism by which trehalose improves cryopreservation outcomes remains not fully understood. Raman microspectroscopy is a powerful tool to provide valuable insight into the nature of interactions among water, trehalose, and Me2SO during cryopreservation. We found that the addition of trehalose to Me2SO based CPA solutions dramatically reduces the area per ice crystals while increasing the number of ice crystals formed when cooled to ?40 or ?80 °C. Differences in ice-formation patterns were found to have a direct impact on cellular viability. Despite the osmotic stress caused by addition of 100 mM trehalose, improvement in cellular viability was observed. However, the substantial increase in osmotic pressure caused by trehalose concentrations above 100 mM may offset the beneficial effects of changing the morphology of the ice crystals achieved by addition of this sugar. |
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| Keywords: | Hepatocellular carcinoma cells Cryopreservation Raman microspectroscopy Respiration Osmotic stress Mitochondria |
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