Cell membrane fluidity and ROS resistance define DMSO tolerance of cryopreserved synovial MSCs and HUVECs

Abstract

Objectives: Synovial mesenchymal stem cells (MSCs) have high freeze–thaw tolerance, whereas human umbilical vein endothelial cells (HUVECs) have low freezing tolerance. The differences in cell type-specific freeze–thaw tolerance and the mechanisms involved are unclear. This study thus aimed to identify the biological and physical factors involved in the differences in freeze–thaw tolerance between MSCs and HUVECs. Materials and methods: For biological analysis, MSC and HUVEC viability after freeze-thawing and alteration of gene expression in response to dimethyl sulfoxide (DMSO, a cryoprotectant) were quantitatively evaluated. For physical analysis, the cell membrane fluidity of MSCs and HUVECs before and after DMSO addition was assessed using a histogram for generalized polarization frequency. Results: HUVECs showed lower live cell rates and higher gene expression alteration related to extracellular vesicles in response to DMSO than MSCs. Fluidity measurements revealed that the HUVEC membrane was highly fluidic and sensitive to DMSO compared to that of MSCs. Addition of CAY10566, an inhibitor of stearoyl-coA desaturase (SCD1) that produces highly fluidic desaturated fatty acids, decreased the fluidity of HUVECs and increased their tolerance to DMSO. The combination of CAY10566 and antioxidant glutathione (GSH) treatment improved HUVEC viability from 57 to 69%. Membrane fluidity alteration may thus contribute to pore-induced DMSO influx into the cytoplasm and reactive oxygen species production, leading to greater cytotoxicity in HUVECs, which have low antioxidant capacity. Conclusions: Differences in freeze–thaw tolerance originate from differences in the cell membranes with respect to fluidity and antioxidant capacity. These findings provide a basis for analyzing cell biology and membrane-physics to establish appropriate long-term preservation methods aimed at promoting transplantation therapies.