Live quantitative monitoring of mineral deposition in stem cells using tetracycline hydrochloride

Macri-Pellizzeri, Laura, De Melo, Nigel, Ahmed, Ifty, Grant, David M., Scammell, Brigitte and Sottile, Virginie (2018) Live quantitative monitoring of mineral deposition in stem cells using tetracycline hydrochloride. Tissue Engineering Part C: Methods, 24 (3). pp. 171-178. ISSN 1937-3384

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The final stage of in vitro osteogenic differentiation is characterized by the production of mineral deposits containing calcium cations and inorganic phosphates, which populate the extracellular matrix surrounding the cell monolayer. Conventional histological techniques for the assessment of mineralization, such as Von Kossa and Alizarin Red S staining, are end-point techniques requiring cell fixation. Moreover, in both cases staining quantitation requires dye extraction which irreversibly alters the ECM conformation and structure, therefore preventing the use of the sample for further analysis.

In this study, the use of Tetracycline hydrochloride (TC) is proposed for the non-destructive staining, quantitation and imaging of mineralizing bone-like nodules in live cultures of human bone marrow mesenchymal stem cells (MSCs) cultured under osteogenic conditions. Overnight administration of TC to living cells was shown not to alter the metabolic activity or the progression of cell differentiation. When applied to differentiating cultures, cell exposure to serial doses of TC was found to produce quantifiable fluorescence emission specifically in osteogenic cultures. Incubation with TC enabled fluorescence imaging of mineralised areas in live cultures and the combination with other fluorophores using appropriate filters. These results demonstrate that serial TC administration over the differentiation time course provides a qualitative and quantitative tool for the monitoring and evaluation of the differentiation process in live cells.

Item Type: Article
Keywords: stem cells, osteogenesis, in vitro differentiation, live cell imaging, quantitative assay
Schools/Departments: University of Nottingham, UK > Faculty of Engineering
University of Nottingham, UK > Faculty of Medicine and Health Sciences > School of Medicine
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Depositing User: Eprints, Support
Date Deposited: 13 Feb 2018 11:13
Last Modified: 08 May 2020 09:30

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