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

Mitsuru Mizuno; Takahisa Matsuzaki; Nobutake Ozeki; Hisako Katano; Hideyuki Koga; Takanori Takebe; Hiroshi Y Yoshikawa; Ichiro Sekiya

doi:10.1186/s13287-022-02850-y

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

Stem Cell Res Ther. 2022 May 3;13(1):177. doi: 10.1186/s13287-022-02850-y.

Authors

Mitsuru Mizuno¹, Takahisa Matsuzaki^{2

3

4}, Nobutake Ozeki⁵, Hisako Katano⁵, Hideyuki Koga⁶, Takanori Takebe^{3

7

8

9

10}, Hiroshi Y Yoshikawa^{2

4

11}, Ichiro Sekiya⁵

Affiliations

¹ Center for Stem Cell and Regenerative Medicine, Tokyo Medical and Dental University (TMDU), 1-5-45, Bunkyo-ku, Yushima, Tokyo, 113-8510, Japan. mizuno.arm@tmd.ac.jp.
² Division of Strategic Research and Development, Graduate School of Science and Engineering, Saitama University, 255, Shimo-okubo, Sakura-ku, Saitama City, Saitama, 338-8570, Japan.
³ Institute of Research, Tokyo Medical and Dental University (TMDU), Tokyo, Japan.
⁴ Department of Applied Physics, Graduate School of Engineering, Osaka University, 2-1, Yamadaoka, Suita City, Osaka, 565-0871, Japan.
⁵ Center for Stem Cell and Regenerative Medicine, Tokyo Medical and Dental University (TMDU), 1-5-45, Bunkyo-ku, Yushima, Tokyo, 113-8510, Japan.
⁶ Department of Joint Surgery and Sports Medicine, Graduate School, Tokyo Medical and Dental University (TMDU), 1-5-45, Bunkyo-ku, Yushima, Tokyo, 113-8519, Japan.
⁷ Organoid Medicine Project, T-CiRA Joint Program, Fujisawa, Kanagawa, Japan.
⁸ Division of Gastroenterology, Hepatology and Nutrition and Division of Developmental Biology, Cincinnati Children's Hospital Medical Center (CCHMC), Cincinnati, OH, USA.
⁹ The Center for Stem Cell and Organoid Medicine (CuSTOM), Cincinnati Children's Hospital Medical Center, Cincinnati, OH, USA.
¹⁰ Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, OH, USA.
¹¹ Department of Chemistry, Saitama University, 255, Shimo-okubo, Sakura-ku, Saitama City, Saitama, 338-8570, Japan.

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.

Keywords: Cell membrane fluidity; Cryopreserve; Human umbilical vein endothelial cells; Mesenchymal stem cells; ROS resistance.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Antioxidants
Cell Membrane / metabolism
Dimethyl Sulfoxide* / pharmacology
Human Umbilical Vein Endothelial Cells / metabolism
Humans
Membrane Fluidity
Mesenchymal Stem Cells* / metabolism
Reactive Oxygen Species / metabolism

Substances

Antioxidants
Reactive Oxygen Species
Dimethyl Sulfoxide