To investigate the effect of hydrogen gas on viability of human mesenchymal stem cell (hMSCs) and human microglial cell line CHME5

Cell viability will be studied with the Cell Titer-Blue assay (Promega Corporation, ). HMSCs or CHME5 cells were seeded in 96-well plates at 5x10³/well in DMEM with 10% FCS in 200 mL volume. Half plates were cultured under normal condition (5% CO2 ambient atmosphere) while another half plated were supplemented with 3.2% hydrogen gas, 5% CO2, balanced air. After 24 h,48h and 72h, the cells were incubated with the cell titer blue reagent for 2 h and then immediately analyzed with SYNERGY 4H reader using an absorbance of 560 nm excitation/590 nm emission reference wavelength (Biotek, Winooski, VT).

After 24h, normal cell fluorescent reading was 41809 ± 660.87AU while MSCs in hydrogen is 8031.0 ± 1274.1 AU ((p<0.0001); after 48h, normal cell fluorescent reading was 22197 ± 448.37 AU while MSCs in hydrogen was 3337.0± 2402.6 AU (p<0.0001);  After 72h, normal cell fluorescent reading is 36116 ± 2981.2 AU while MSCs in hydrogen is 1461.2 ± 704.53 AU (p=0.0079). Similar data were obtained with CHME5.

These data suggest that hydrogen gas have obvious toxicity effect on cell viability of hMSCs and CHME5 cells, which may raise some questions about the clinical application of hydrogen gas. The specific mechanism is still under investigation