Optimization of differential filtration-based mitochondrial isolation for mitochondrial transplant to cerebral organoids

Stem Cell Research & Therapy

Research

Liliana Attisano¹  Douglas C. Wallace²  Hyunjin Jeong³  David F. Bodenstein³  Kassandra A. Zachos³  Pavel Powlowski³  Dana El Soufi El Sabbagh³  Landon Edgar⁴  Ana Cristina Andreazza⁵ 

[1] Department of Biochemistry, University of Toronto, Toronto, ON, Canada;Donnelly Centre, University of Toronto, Toronto, ON, Canada;Department of Biological Chemistry, Center for Molecular and Mitochondrial Medicine and Genetics, University of California, Irvine, CA, USA;Division of Human Genetics, Department of Pediatrics, Center for Mitochondrial and Epigenomic Medicine, Children’s Hospital of Philadelphia, Philadelphia, USA;Department of Pediatrics, The Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA;Department of Pharmacology and Toxicology, University of Toronto, Medical Science Building, Room 4211, 1 King’s College Circle, M5S 1A8, Toronto, ON, Canada;Department of Pharmacology and Toxicology, University of Toronto, Medical Science Building, Room 4211, 1 King’s College Circle, M5S 1A8, Toronto, ON, Canada;Department of Chemistry, University of Toronto, Toronto, ON, Canada;Department of Pharmacology and Toxicology, University of Toronto, Medical Science Building, Room 4211, 1 King’s College Circle, M5S 1A8, Toronto, ON, Canada;Department of Psychiatry, University of Toronto, Toronto, ON, Canada;

关键词: Mitochondria;    Mitochondrial isolation;    Mitochondrial transplant;    Induced pluripotent stem cells;    Cerebral organoids;   

DOI  :  10.1186/s13287-023-03436-y

 received in 2022-11-28, accepted in 2023-07-27,  发布年份 2023

来源: Springer

【 摘 要 】

BackgroundMitochondrial 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.MethodsTherefore, 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.ResultsWe 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.ConclusionsThus, 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.

【 授权许可】

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

【 图 表 】

13690_2023_1147_Article_IEq18.gif

Fig. 1

Fig. 2

Fig. 1

Fig. 5

Fig. 1