线粒体功能失调参与到从遗传性线粒体疾病到慢性代谢性疾病的几种疾病之中。一种新兴的治疗线粒体功能失调的方法是移植自体活体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，并在线粒体分离样品中发现了整个细胞的杂质。因此，我们探索了用于多种细胞系（包括HEK293细胞和诱导多能干细胞（iPSCs））的5μm滤膜（滤膜A和滤膜B）的替代型线粒体分离方法。使用MitoTracker™染色和流式细胞术，对可行线粒体的浓度进行了定量。我们通过将mitoDsRed2标记的线粒体转移入H9源性脑器官样体，进行了线粒体移植的验证。我们发现，相较于原始方案中使用的5μm滤膜，滤膜B提供了最高质量的线粒体。利用这种方法，我们还成功地从诱导多能干细胞中分离了线粒体。为了测试可行性，我们分离并将mitoDsRed2标记的线粒体移植到H9源性脑器官样体中，并通过TOMM20和MAP2的免疫荧光共局化来观察线粒体是否被吞噬。因此，在差异过滤方法中使用滤膜B是从细胞中分离纯净且可行的线粒体的理想选择，这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。© 2023 BioMed Central有限公司，施普林格自然出版集团的一部分。

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.© 2023. BioMed Central Ltd., part of Springer Nature.