Beijing Institute of Ophthalmology, Beijing Tongren Eye Center, Beijing Tongren Hospital, Capital Medical University.
Beijing Institute of Ophthalmology, Beijing Tongren Eye Center, Beijing Tongren Hospital, Capital Medical University;
J Vis Exp. 2024 Oct 4(212). doi: 10.3791/66917.
Mitochondrial transfer is a normal physiological phenomenon that occurs widely among various types of cells. In the study to date, the most important pathway for mitochondrial transport is through tunneling nanotubes (TNTs). There have been many studies reporting that mesenchymal stem cells (MSCs) can transfer mitochondria to other cells by TNTs. However, few studies have demonstrated the phenomenon of bidirectional mitochondrial transfer. Here, our protocol describes an experimental approach to study the phenomenon of mitochondrial transfer between MSCs and retinal pigment epithelial cells in vitro by two mitochondrial tracing methods. We co-cultured mito-GFP-transfected MSCs with mito-RFP-transfected ARPE19 cells (a retinal pigment epithelial cell line) for 24 h. Then, all cells were stained with phalloidin and imaged by confocal microscopy. We observed mitochondria with green fluorescence in ARPE19 cells and mitochondria with red fluorescence in MSCs, indicating that bidirectional mitochondrial transfer occurs between MSCs and ARPE19 cells. This phenomenon suggests that mitochondrial transport is a normal physiological phenomenon that also occurs between MSCs and ARPE19 cells, and mitochondrial transfer from MSCs to ARPE19 cells occurs much more frequently than vice versa. Our results indicate that MSCs can transfer mitochondria into retinal pigment epithelium, and similarly predict that MSCs can fulfill their therapeutic potential through mitochondrial transport in the retinal pigment epithelium in the future. Additionally, mitochondrial transfer from ARPE19 cells to MSCs remains to be further explored.
线粒体转移是一种广泛存在于各种类型细胞中的正常生理现象。在迄今为止的研究中，线粒体运输最重要的途径是通过隧道纳米管（TNTs）。有许多研究报告称，间充质干细胞（MSCs）可以通过 TNTs 将线粒体转移到其他细胞中。然而，很少有研究表明存在双向线粒体转移。在这里，我们的方案描述了一种通过两种线粒体示踪方法在体外研究 MSCs 和视网膜色素上皮细胞之间线粒体转移现象的实验方法。我们将 mito-GFP 转染的 MSCs 与 mito-RFP 转染的 ARPE19 细胞（视网膜色素上皮细胞系）共培养 24 小时。然后，所有细胞均用鬼笔环肽染色，并通过共聚焦显微镜成像。我们观察到 ARPE19 细胞中有绿色荧光的线粒体和 MSCs 中有红色荧光的线粒体，这表明 MSCs 和 ARPE19 细胞之间发生了双向线粒体转移。这一现象表明，线粒体运输是一种正常的生理现象，也发生在 MSCs 和 ARPE19 细胞之间，而且线粒体从 MSCs 转移到 ARPE19 细胞比反之更为频繁。我们的结果表明 MSCs 可以将线粒体转移到视网膜色素上皮细胞中，并且同样可以预测，MSCs 可以通过未来在视网膜色素上皮细胞中的线粒体运输来实现其治疗潜力。此外，来自 ARPE19 细胞的线粒体向 MSCs 的转移仍有待进一步探索。
