Live Quantitative Monitoring of Mineral Deposition in Stem Cells Using Tetracycline Hydrochloride.
Live Quantitative Monitoring of Mineral Deposition in Stem Cells Using Tetracycline Hydrochloride.
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DOI：
10.1089/ten.tec.2017.0400
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发表时间：
2018-03
期刊：
影响因子：
--
通讯作者：
Sottile V
中科院分区：
文献类型：
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作者：
Macri-Pellizzeri L;De Melo N;Ahmed I;Grant D;Scammell B;Sottile V
体外成骨分化的最后阶段是产生含有钙离子和无机磷酸盐的矿物质，这些矿物质充填在细胞单层周围的细胞外基质(ECM)中。评估矿化的传统组织学技术，如冯·科萨和茜素红S染色，是需要细胞固定的终点技术。此外，在这两种情况下，染色定量都需要提取染料，这将不可逆转地改变ECM的构象和结构，因此无法使用样品进行进一步分析。在这项研究中，四环素盐酸盐(TC)用于在成骨条件下培养的人骨髓间充质干细胞的活培养中对矿化骨样结节进行无损染色、定量和成像。过夜给活细胞的TC被证明不会改变代谢活动或细胞分化的进程。当应用于分化培养时，细胞暴露于连续剂量的TC被发现产生可量化的荧光发射，特别是在成骨培养中。与TC孵育能够对活培养物中的矿化区域进行荧光成像，并使用适当的过滤器与其他荧光团结合。这些结果表明，在分化过程中连续给予TC为监测和评估活细胞的分化过程提供了一种定性和定量的工具。
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 (ECM) 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 nondestructive staining, quantitation, and imaging of mineralizing bone-like nodules in live cultures of human bone marrow mesenchymal stem cells 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 mineralized 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.