| Summary: | 碩士 === 國立臺灣大學 === 化學研究所 === 99 === Fluorescent nanodiamond (FND) is a sp3-carbon-based nanomaterial produced by radiation-damage followed by annealing. FND possess several unique properties such as excellent biocompatibility, facile surface modification, high tissue-penetrable red fluorescence and outstanding photostability, making it well suited for long-term labeling and tracking of cells. In this study, we use surface-oxidized FND particles (size~100nm) as the cell tracker. We introduce FNDs into cells by endocytosis through incubation, and do flow cytometric analysis. Based on different cell types, the thesis could be divided into two parts.
In the first part, we label 3T3-L1 pre-adipocytes and 489-2.1 multipotential stromal cells with FNDs, and use carboxy-fluorescein diacetate succinimidyl ester (CFSE) for comparison. Flow cytometric analysis reveals that the mean fluorescence intensity of the ingested FND particles per cell decays nearly exponentially with the number of cell division. The observed half-life matches well with the cell doubling time within 10%. Since the fluorescence emission of FNDs is perfectly stable, one can take advantage of this feature and determine conversely the number of generation that the cells have gone through by measuring their fluorescence intensity profile. In this study, we track to the seventh cell division. Also, we demonstrate for the first time that high-purity sorting of FND-labeled cells in a 1:1000 cell mixture is achievable by utilizing both the fluorescence and light scattering properties of this novel nanomaterial. Finally, we assessed the effect of the FND labeling and cell sorting on the 489-2.1 cells by using the cell proliferation assay. No significant difference in cell proliferation of FND-labeled cells before and after sorting was found. It signifies that fluorescence activated cell sorting (FACS) and the FND labeling do not alter the cell proliferation of the multipotential stromal cells.
In the second part, we demonstrate for the first time the application of FND to the primary cells. After labeling the lung stem cells directly isolated from mice with FNDs, we introduce those cells through intravenous injection. By flow cytometric analysis and microscopy, we find that the lung stem cells would reside particularly in the lung. We also use immunohistochemistry (IHC) to prove that the lung stem cells are not engulfed by macrophages. By combining the results in this study, we provide a convincing evidence that the lung stem cells do have homing ability.
In this study, we build up a biocompatible nanoparticle platform that combines FND labeling, FACS and microscopy. It had already begun to advance its ability to do long-term in vivo stem cell tracking research.
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