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Updated: Jul 19, 2025
Rapid Isolation And Purification Of Mitochondria For Transplantation By Tissue Dissociation And Differential Filtration
Published on: September 6, 2014
Optimization of differential filtration-based mitochondrial isolation for mitochondrial transplant to cerebral
David F Bodenstein1, Pavel Powlowski1, Kassandra A Zachos1
1Department of Pharmacology and Toxicology, University of Toronto, Medical Science Building, Room 4211, 1 King's College Circle, Toronto, ON, M5S 1A8, Canada.
Background:
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.
Methods:
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.
Results:
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.
Conclusions:
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.
Insights
Researchers optimized mitochondrial isolation for cell therapy. Using filter B, they successfully purified viable mitochondria from various cell types, including stem cells, paving the way for safe mitochondrial transplantation.
Area of Science:
- Mitochondrial biology
- Cellular therapy
- Regenerative medicine
Background:
- Mitochondrial dysfunction contributes to various diseases.
- Autologous live mitochondria transplantation is an emerging therapeutic strategy.
- Previous isolation protocols yielded contaminated mitochondrial samples.
Purpose of the Study:
- To optimize mitochondrial isolation for cellular models.
- To identify superior filters for isolating pure, viable mitochondria.
- To evaluate the feasibility of mitochondrial transplantation in organoids.
Main Methods:
- Tested alternative 5-μm filters (A and B) for mitochondrial isolation.
- Utilized MitoTracker™ staining and flow cytometry for quantification.
- Performed proof-of-principle mitochondrial transplants into cerebral organoids.
Main Results:
- Filter B yielded higher quality mitochondria compared to the original protocol's filter.
- Successfully isolated viable mitochondria from HEK293 cells and induced pluripotent stem cells (iPSCs).
- Demonstrated mitochondrial engraftment in cerebral organoids via immunofluorescence.
Conclusions:
- Filter B is optimal for isolating pure, viable mitochondria using differential filtration.
- This method enables further research into mitochondrial transplantation safety and integration.
- Optimized isolation supports the advancement of cell-based mitochondrial therapies.
