Sogawa Koushirou, Okawa Ryoma, Yachiku Kenji, Shiozaki Motoko, Miura Takanori, Takayanagi Hiroshi, Shibata Takashi, Ezoe Sachiko
Department of Environmental Space Infection Control, Graduate School of Medicine/Faculty of Medicine, Osaka University, 1-3 Yamadaoka, Suita, Osaka, 565-0871, Japan.
Department of Ophthalmology, Graduate School of Medicine, Osaka University, 2-2 Yamadaoka, Suita, Osaka, 565-0871, Japan.
Regen Ther. 2020 Feb 24;14:184-190. doi: 10.1016/j.reth.2019.12.007. eCollection 2020 Jun.
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.
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.
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.
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.
卫生管理在细胞产品制造中比在化学制剂生产中更为重要，因为细胞材料和最终产品无法进行去污处理。另一方面，尤其是在选择卫生制剂时，必须考虑其对细胞的不利影响以及去污效果。二氧化氯（ClO）是一种强效消毒剂，目前有望成为细胞生产领域安全有效的卫生制剂。在本研究中，我们调查了二氧化碳培养箱环境中低剂量ClO气体对培养于其中的间充质干细胞（MSCs）特性的影响。
首先，我们在用于细胞培养的二氧化碳培养箱中安装了一个ClO发生器，可维持恒定水平的ClO。在二氧化碳培养箱中培养人脐带来源的MSCs后，对细胞特性进行分析。
持续暴露于0.05 ppmv的ClO气体至少到第8代时都不影响细胞增殖。在荧光激活细胞分选（FACS）分析中，通常在MSCs上表达的抗原CD105、CD90、CD44、CD73和CD29呈阳性观察结果，但分化标志物CD11b和CD34在暴露于0.05 ppmv或0.1 ppmv ClO气体的MSCs上表达很少，与对照细胞情况相同。在细胞死亡调查中，0.05 ppmv和0.1 ppmv的ClO气体对MSCs的活力、凋亡或坏死影响很小。此外，我们使用衰老相关β - 半乳糖苷酶（SA - β - gal）染色评估衰老情况。虽然在0.1 ppmv ClO气体中培养的染色细胞频率比未暴露细胞显著增加，但在0.05 ppmv中染色细胞很少，其频率与对照几乎相同。
所有这些结果表明，虽然过高浓度的ClO气体会诱导衰老，但既不诱导凋亡也不诱导细胞分化，暴露于0.05 ppmv的ClO气体对MSCs特性影响很小。在本研究中我们证明，持续暴露于适当剂量的ClO气体可安全用作细胞处理设施中的去污剂。
