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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; Sottile, Virginie

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Authors

Nigel De Melo

DAVID GRANT DAVID.GRANT@NOTTINGHAM.AC.UK
Professor of Materials Science

Brigitte Scammell

Virginie Sottile



Abstract

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.

Citation

Macri-Pellizzeri, L., De Melo, N., Ahmed, I., Grant, D. M., Scammell, B., & Sottile, V. (2018). Live quantitative monitoring of mineral deposition in stem cells using tetracycline hydrochloride. Tissue Engineering Part C Methods, 24(3), https://doi.org/10.1089/ten.TEC.2017.0400

Journal Article Type Article
Acceptance Date Jan 4, 2018
Online Publication Date Feb 27, 2018
Publication Date Mar 30, 2018
Deposit Date Feb 13, 2018
Publicly Available Date Mar 28, 2024
Journal Tissue Engineering Part C: Methods
Electronic ISSN 1937-3384
Publisher Mary Ann Liebert
Peer Reviewed Peer Reviewed
Volume 24
Issue 3
DOI https://doi.org/10.1089/ten.TEC.2017.0400
Keywords stem cells, osteogenesis, in vitro differentiation, live cell imaging, quantitative
assay
Public URL https://nottingham-repository.worktribe.com/output/922205
Publisher URL https://www.liebertpub.com/doi/10.1089/ten.tec.2017.0400

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