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

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: Feb 27, 2018
• 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
• Contract Date: Feb 13, 2018