Mitochondria transplantation between living cells

Abstract

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.

Engineering Endosymbiotic Growth of E. coli in Mammalian Cells

“…enterica as a positive control because the bacterium is known for its ability to grow in the cytosol of mammalian cells . To enable fluidic handling and injection of the bacteria, we used cylindrical FluidFM probes . To freely move within the microfluidic channel of the cantilevers, the minimal Feret diameter of the bacteria needs to be smaller than the channel dimensions of the probe (Figure A).…”

“… 18 To enable fluidic handling and injection of the bacteria, we used cylindrical FluidFM probes. 19 To freely move within the microfluidic channel of the cantilevers, the minimal Feret diameter of the bacteria needs to be smaller than the channel dimensions of the probe ( Figure 1 A). We therefore measured the diameters of the chosen bacteria ( Figure 1 B) and found that E. coli , V. natriegens, and S. enterica can be handled with a channel width of 1.2 μm, while A. vinelandii requires a larger channel width of 1.6 μm ( Figure 1 C).…”

“…In this study, we introduce FluidFM as an efficient tool to inject bacteria into cultured cells while preserving viability of both organisms. Recently developed FluidFM cantilevers with cylindrical apexes 19 allow injection of diverse bacteria. We deduced intracellular growth from individual injected host cells using diverse fluorescently labeled bacteria.…”

“…Cylindrical FluidFM-probes were prepared as in Gäbelein et al. 19 The used protocol only deviates in the used FIB-scanning electron microscopy (SEM) imaging setup: for the current paper, a Helios 5UX DualBeam FIB-SEM setup (ThermoFisher, USA) was used. The image in Figure 1 A was taken using FIB-imaging.…”

“…The workflow presented in this work was optimized for cultured cells under a 63× oil objective (see “FluidFM setup and microscopy” above). Host cell viability post injection depends on the injected volume, which can be tuned by setting the positive pressure value of the fluidic system and the time of injection, as previously characterized for this probe type 19 and other FluidFM probes. 17 Following injection, bacteria only start to proliferate in a subset of injected cells, these cells were subsequently used to quantify the doubling time.…”