Department of Pharmacology and Toxicology, University of Toronto, Medical Science Building, Room 4211, 1 King's College Circle, Toronto, ON, M5S 1A8, Canada.
Department of Biochemistry, University of Toronto, Toronto, ON, Canada.
Stem Cell Res Ther. 2023 Aug 15;14(1):202. doi: 10.1186/s13287-023-03436-y.
Mitochondrial dysfunction is involved in several diseases ranging from genetic mitochondrial disorders to chronic metabolic diseases. An emerging approach to potentially treat mitochondrial dysfunction is the transplantation of autologous live mitochondria to promote cell regeneration. We tested the differential filtration-based mitochondrial isolation protocol established by the McCully laboratory for use in cellular models but found whole cell contaminants in the mitochondrial isolate.
Therefore, we explored alternative types of 5-μm filters (filters A and B) for isolation of mitochondria from multiple cell lines including HEK293 cells and induced pluripotent stem cells (iPSCs). MitoTracker™ staining combined with flow cytometry was used to quantify the concentration of viable mitochondria. A proof-of-principle mitochondrial transplant was performed using mitoDsRed2-tagged mitochondria into a H9-derived cerebral organoid.
We found that filter B provided the highest quality mitochondria as compared to the 5-μm filter used in the original protocol. Using this method, mitochondria were also successfully isolated from induced pluripotent stem cells. To test for viability, mitoDsRed2-tagged mitochondria were isolated and transplanted into H9-derived cerebral organoids and observed that mitochondria were engulfed as indicated by immunofluorescent co-localization of TOMM20 and MAP2.
Thus, use of filter B in a differential filtration approach is ideal for isolating pure and viable mitochondria from cells, allowing us to begin evaluating long-term integration and safety of mitochondrial transplant using cellular sources.
线粒体功能障碍与多种疾病有关，从遗传性线粒体疾病到慢性代谢性疾病。一种有潜力治疗线粒体功能障碍的新兴方法是移植自体活线粒体以促进细胞再生。我们测试了 McCully 实验室建立的基于差异过滤的线粒体分离方案，用于细胞模型，但在线粒体分离物中发现了完整细胞污染物。
因此，我们探索了替代类型的 5μm 过滤器（过滤器 A 和 B），用于分离包括 HEK293 细胞和诱导多能干细胞（iPSC）在内的多种细胞系中的线粒体。使用 MitoTracker™染色结合流式细胞术来定量活线粒体的浓度。使用 mitoDsRed2 标记的线粒体进行了线粒体移植的原理验证实验，将其移植到 H9 衍生的类器官中。
与原始方案中使用的 5μm 过滤器相比，我们发现过滤器 B 提供了质量最高的线粒体。使用这种方法，也成功地从诱导多能干细胞中分离出线粒体。为了测试线粒体的活力，分离并移植了 mitoDsRed2 标记的线粒体到 H9 衍生的类器官中，并观察到 TOMM20 和 MAP2 的免疫荧光共定位表明线粒体被吞噬。
因此，在差异过滤方法中使用过滤器 B 是从细胞中分离纯净和有活力的线粒体的理想选择，使我们能够开始评估使用细胞来源的线粒体移植的长期整合和安全性。
