Important methodological aspects that should be taken into account during the research of isolated mitochondria

Rendón, Dairo Alonso

doi:10.1016/j.ab.2019.113492

“…The different success rates between the 2 alternative protocols can be explained by differences in mitochondrial condition. When evaluating mitochondrial extraction protocols, we observed that a fraction of the extracted mitochondria undergo rupture of their outer membranes ( S7 Fig ) [ 31 ]. Irreversible damage of mitochondria leads to degradation inside cells and potentially cell apoptosis due to cytochrome c leakage.…”

Section: Resultsmentioning

confidence: 99%

Mitochondria transplantation between living cells

Gäbelein

¹

,

Feng

²

,

Sarajlic³

et al. 2022

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Mitochondria and the complex endomembrane system are hallmarks of eukaryotic cells. To date, it has been difficult to manipulate organelle structures within single live cells. We developed a FluidFM-based approach to extract, inject, and transplant organelles from and into living cells with subcellular spatial resolution. The technology combines atomic force microscopy, optical microscopy, and nanofluidics to achieve force and volume control with real-time inspection. We developed dedicated probes that allow minimally invasive entry into cells and optimized fluid flow to extract specific organelles. When extracting single or a defined number of mitochondria, their morphology transforms into a pearls-on-a-string phenotype due to locally applied fluidic forces. We show that the induced transition is calcium independent and results in isolated, intact mitochondria. Upon cell-to-cell transplantation, the transferred mitochondria fuse to the host cells mitochondrial network. Transplantation of healthy and drug-impaired mitochondria into primary keratinocytes allowed monitoring of mitochondrial subpopulation rescue. Fusion with the mitochondrial network of recipient cells occurred 20 minutes after transplantation and continued for over 16 hours. After transfer of mitochondria and cell propagation over generations, donor mitochondrial DNA (mtDNA) was replicated in recipient cells without the need for selection pressure. The approach opens new prospects for the study of organelle physiology and homeostasis, but also for therapy, mechanobiology, and synthetic biology.

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“…The different success rates between the two alternative protocols can be explained by differences in mitochondrial condition. When evaluating mitochondrial extraction protocols, we observed that a fraction of the extracted mitochondria undergo rupture of their outer membranes (Supplementary Fig 8) (32). Irreversible damage of mitochondria leads to degradation inside cells and potentially cell apoptosis due to cytochrome c leakage.…”

Section: Mitochondrial Transplantation Into Cultured Cellsmentioning

confidence: 99%

Mitochondria transplantation between living cells

Gäbelein

¹

,

Feng

²

,

Sarajlic³

et al. 2021

Preprint

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Mitochondria and the complex endomembrane system are hallmarks of eukaryotic cells. To date, it has been difficult to manipulate organelle structures within single live cells. We developed a FluidFM-based approach to extract, inject and transplant organelles from and into living cells with subcellular spatial resolution. The approach enabled the transfer of controlled quantities of mitochondria into cells while maintaining their viability and monitoring their fate in new host cells. Transplantation of healthy and drug-impaired mitochondria into primary keratinocytes allowed real-time tracking of mitochondrial subpopulation rescue. Fusion with the mitochondrial network of recipient cells occurred 20 min after transplantation and continued for over 16 hours. After transfer of mitochondria and cell propagation over generations, we show that donor mtDNA was replicated in recipient cells without the need for selection pressure. The approach opens new prospects for the study of organelle physiology and homeostasis, but also for mechanobiology, synthetic biology, and therapy.

show abstract