OPTIMIZATION OF COBALT CHLORIDE CONCENTRATION FOR INDUCING HYPOXIA-ASSOCIATED CELLULAR RESPONSES IN FIBROBLASTS

Abstract

Introduction: The objective of this study was to investigate the optimal concentration of cobalt chloride for inducing hypoxia-mimicking conditions in human gingival fibroblast (HGFs) and L929 fibroblast cell lines. Methods: HGFs and L929 were induced into hypoxia-mimic conditions using cobalt chloride (CoCl₂). The cytotoxicity of CoCl₂ was evaluated using the PrestoBlue assay at concentrations of 0, 50, 100, 150, and 200 µM over 12, 24, 48, 72, and 96 hours. Optical density was measured and analysed using one-way ANOVA. Hypoxia-induced cellular responses were assessed by measuring fluorescence intensity. Cells treated with 200 µM CoCl₂ were incubated with Photo-oxidation Resistant 2’,7’-Dichlorodihydrofluorescein diacetate (DCFH-DA) and fluorescence intensity was measured at 24, 72, and 120 hours. Results: In HGFs, exposure to 200 µM CoCl₂ significantly reduced cell viability compared with the control and other concentrations at 48, 72, and 96 hours. In L929 cells, 200 µM CoCl₂ significantly decreased cell viability at 96 hours compared to lower concentrations. DCFH-DA fluorescence intensity increased in both HGFs and L929 cells treated with 200 µM CoCl₂, compared to controls at 24, 72, and 120 hours. Conclusion: These findings indicate that 200 µM CoCl₂ induces oxidative stress and cytotoxic effects in both cell types. These responses are consistent with cellular stress conditions, including those observed under hypoxia-related environments, and may be relevant to hypoxia-associated cellular responses.

Keywords: Fibroblast, Hypoxia, Cobalt Chloride