Stable transplantation of human mitochondrial DNA by high-throughput, pressurized mitochondrial delivery

Generating mammalian cells with specific mtDNA-nDNA combinations is desirable but difficult to achieve and would be enabling for studies of mitochondria-nucleus communication and coordination in controlling cell fates and functions. We developed ‘MitoPunch’, a force-actuated mitochondrial transfer device, to deliver isolated mitochondria into numerous target mammalian cells simultaneously. MitoPunch and MitoCeption, an alternative force-based mitochondrial transfer approach, both yield stable isolated mitochondrial recipient (SIMR) cells that permanently retain exogenous mtDNA, whereas coincubation of mitochondria with cells does not yield SIMR cells. Although a typical MitoPunch or MitoCeption delivery results in dozens of immortalized SIMR clones with restored oxidative phosphorylation, only MitoPunch can produce replication-limited, non-immortal human SIMR clones. The MitoPunch device is versatile, inexpensive to assemble, and easy to use for engineering mtDNA-nDNA combinations to enable fundamental studies and potential translational applications.

MitoCeption only generated clones in 143BTKρ 0 cells and was unable to form stable 1 1 clones in replication-limited BJ cells.

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We next investigated whether differences in SIMR clone generation between 1 3 143BTKρ 0 and BJ ρ 0 cells is driven by sensitivity to differences in delivery pressure. 1 4 We developed a MitoPunch device with adjustable plunger acceleration modulated by 1 5 changing the circuit voltage. We achieved maximum 143BTKρ 0 SIMR clone 1 6 generation with this tunable MitoPunch at 1 V, with a sharp reduction to background 1 7 with increasing voltage ( Figure 3D). The BJ ρ 0 recipient also showed maximal SIMR 1 8 generation at 1 V, with a shallow decline in SIMR generation efficiency to 5 V. tested, showing that optimal mitochondrial delivery pressure may be cell type 2 2 dependent ( Figure S2A). We performed a similar force titration with MitoCeption by 2 3 8 adjusting the maximum centripetal force. In 143BTKρ 0 cells, we observed maximum 1 clone generation at 1000 x g and we obtained no BJ ρ 0 SIMR clones at any 2 acceleration tested for MitoCeption ( Figure 3E). These data suggest that MitoPunch is 3 uniquely able to generate SIMR clones in replication-limited fibroblasts and SIMR 4 generation efficiency depends on delivery pressure.

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To enable desirable mtDNA-nDNA clone generation using limited starting 6 material, such as mitochondria from rare cell subpopulations, we determined the 7 minimal biomass of mitochondrial isolate required to generate SIMR clones. We concentrations. After delivery, we seeded half of each sample for SIMR cell selection in 1 1 uridine-free medium and analyzed the fraction of cells that received mitochondria in the MitoPunch, so we tested whether the same sample could be used for serial transfers.

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We performed 11 sequential deliveries into 143BTKρ 0 cells using one aliquot of 2 3 9 mitochondrial isolate and found maximal SIMR clone generation from the first and 1 second deliveries, after which SIMR cell formation dropped off by the 11 th transfer 2 ( Figure S2C). culture passaging, we grew four clones, two from MitoPunch and two from MitoCeption, 1 2 through two freeze/thaw cycles and measured their respiration. We found that one 1 3 MitoCeption clone lost its respiratory capacity and one MitoPunch clone was not viable 1 4 after freeze down and thaw (data not shown). In the remaining clones, basal and 1 5 maximal respiration, spare respiratory capacity, and ATP generation remained stable 1 6 throughout both freeze thaw cycles ( Figure 4A). We then imaged the SIMR clones for 1 7 TOM20 mitochondrial outer membrane protein and double-stranded DNA levels by and metabolome of replication-limited SIMR clones differs significantly from un-1 2 manipulated control clones but can be recovered and reset to un-manipulated control   identified these cell lines, however, we have sequenced their mitochondrial and nuclear 1 8 DNA for polymorphisms and find unique sequences which we use for genotyping our 1 9 cultures (unpublished data).  Mitochondria isolated from HEK293T dsRed cells re-suspended in 1x DPBS with 1 1 calcium and magnesium at ~1mg total protein/ml were pipetted on top of the culture  The construction and actuation of the MitoPunch device is described in more detail in  addition, a prototype based on the same principles with identical delivery procedures as 1 5 MitoPunch, but with tunable plunger force achieved by varying actuator voltage, was 1 6 engineered by NanoCav, LLC. suspended in 1x DPBS with calcium and magnesium at ~1mg total protein/ml was 2 2 pipetted into the well and the plate was centrifuged at 1500 x g for 15 min at 4°C. Cells 2 3 14 were removed from the centrifuge and incubated for 2 h at 37°C and 5% CO 2 before 1 being centrifuged a second time at 1500 x g for 15 min at 4°C. Cells were then released 2 from the dish and placed into 10 cm plates for SIMR cell selection or harvested for 3 additional analyses.  was approximated by plates (~36 h in dialyzed FBS galactose medium), the medium was changed to uridine-2 0 free medium and colonies were counted by microscopy or isolated using cloning rings.  Confocal microscopy 1 1 Cells (1 x 10 5 ) were plated in 6-well dishes with 2 ml of media on glass coverslips 1x PBS, pH 7.4, was pipetted onto samples and incubated for 15 min at RT.

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Paraformaldehyde was aspirated and samples were washed 3x with 1x PBS, pH 7.4.

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Samples were further washed with PBS 3x with 5 min RT incubation per wash. incubations and mounted on microscope slides.

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To mount, samples were removed from the 6-well dish and rinsed by dipping in 1 4 deionized water, dried with a Kimwipe, and mounted using ProLong Gold Antifade Media was aspirated from 10 cm plates before fixation with 1 ml freshly diluted 4% violet was removed and plates were washed 2x with deionized water before drying 1 7 overnight at RT and visual quantification. Oxygen consumption rate measurements 2 0 Oxygen consumption rate (OCR) measurements used a Seahorse XF96 Extracellular Flux Analyzer (Agilent, Santa Clara, CA). 2 x 10 4 cells were seeded into each well of a 2 2 V3 96-well plate (Agilent, Cat. # 101085-004) and cultured 24 h before measuring OCR. The Agilent Seahorse mitochondrial stress test was used to quantify OCR for basal 1 respiration and respiration following the sequential addition of mitochondrial inhibitors 2 oligomycin, carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP), and 3 rotenone. transfer. MitoCeption and coincubation were carried out as described above, and 1 0 MitoPunch was performed with PI FluorBrite medium loaded into the PDMS reservoir 1 1 and incubated for 15 min at 37°C and 5% CO 2 . All samples were washed with 1x PBS Quantification and statistical analysis 1 8 All information pertaining to experimental replication is found in the figure legends. All