Effects of continuous exposure to low concentration of ClO gas on the growth, viability, and maintenance of undifferentiated MSCs in long-term cultures.

Introduction: Hygienic management is more important in the manufacturing of cell products than in the production of chemical agents, because cell material and final product cannot be decontaminated. On the other hand, especially in the selection of hygienic agent, the adverse effects on the cells must be considered as well as the decontamination effect. ClO is a potent disinfectant, which is now expected as a safe and effective hygienic agent in the field of cell production. In this study, we investigated the effects of low dose ClO gas in the atmosphere of CO incubator on the characteristics of MSCs cultured in it.

Methods: First, we installed a ClO generator to a CO incubator for cell culture in which a constant level of ClO can be maintained. After culturing human cord derived MSCs in the CO incubator, the characteristics of cells were analyzed.

Results: Continuous exposure to 0.05 ppmv of ClO gas did not affect cell proliferation until at least 8th passage. In the FACS analysis, antigens usually expressed on MSCs, CD105, CD90, CD44, CD73 and CD29, were positively observed, but differentiation markers, CD11b and CD34, were little expressed on the MSCs exposed to 0.05 ppmv or 0.1 ppmv of ClO gas just as on the control cells. Also in the investigation for cell death, 0.05 ppmv and 0.1 ppmv of ClO gas little affected the viability, apoptosis or necrosis of MSCs. Furthermore, we assessed senescence using SA-β-gal staining. Although the frequency of stained cells cultured in 0.1 ppmv of ClO gas was significantly increased than that of not exposed cells, the stained cells in 0.05 ppmv were rare and their frequency was almost the same as that in control.

Conclusions: All these results indicate that, although excessive concentration of ClO gas induces senescence but neither apoptosis nor cell differentiation, exposure to 0.05 ppmv of ClO gas little affected the characteristics of MSCs. In this study we demonstrate that continuous exposure to appropriate dose of ClO gas can be safely used as decontamination agent in cell processing facilities.