Hydrogen gas improves proliferation and mitochondrial activity of human adipose-derived stem cells after cryopreservation

Abstract

The objective of this study is to evaluate the effect of hydrogen gas on the biological functions of human adipose-derived stem cells (hADSC) in cryopreservation. hADSC were cryopreserved by a commercial cell preservation solution in the presence of hydrogen gas. After cryopreservation at 80 C, the viability, initial attachment morphology, and biological parameters of cells cryopreserved were evaluated to compare with those of cells cryopreserved in the absence of hydrogen gas. The hydrogen concentration in the cell preservation solution was 2.0 ppm immediately after preparation and after that decreased with time. The presence of hydrogen gas permitted cells to significantly increase the proliferation of cells in addition to the percent initial adhesion. The number of cells in the spread state was significantly high compared with that of hydrogen gas-free cryopreserved cells. The cell cycle measurement with the flow cytometry and measurement of intracellular reactive oxygen species (ROS) were performed to demon- strate an enhanced cell cycle and a decreased ROS production. In the cell cycle assay, the percentage of cells in the mitotic phase increased. The presence of hydrogen gas decreased hydroxyl radicals imme- diately to a significantly great extent after thawing. It is concluded that the presence of hydrogen gas during cryopreservation is promising to improve the biological behavior of cells after cell thawing in terms of cells viability, proliferation or metabolic activity. © 2024 The Author(s). Published by Elsevier BV on behalf of The Japanese Society for Regenerative Medicine. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Study details

Article type

Original Research

Dose comparison

Hydrogen concentration in the preservation solution was measured (1.2 ppm used for main experiments), and effects were compared to hydrogen-free controls.

Dose-dependent effect

Yes

Outcome

Positive

Findings

Hydrogen gas during cryopreservation of human adipose-derived stem cells (hADSC) significantly improved cell proliferation, initial adhesion, cell cycle progression, and mitochondrial activity after thawing, while reducing intracellular ROS and hydroxyl radical levels. No significant difference in cell viability was observed, but hydrogen gas enhanced biological functions and morphology post-thaw.