1. Cell Biology

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

  1. Alexander J Sercel
  2. Alexander N Patananan
  3. Tianxing Man
  4. Ting-Hsiang Wu
  5. Amy K Yu
  6. Garret W Guyot
  7. Shahrooz Rabizadeh
  8. Kayvan R Niazi
  9. Pei-Yu Chiou
  10. Michael A Teitell  Is a corresponding author
  1. Molecular Biology Interdepartmental Doctoral Program, University of California, Los Angeles, United States
  2. Department of Pathology and Laboratory Medicine, David Geffen School of Medicine, University of California, Los Angeles, United States
  3. Department of Mechanical and Aerospace Engineering, University of California, Los Angeles, United States
  4. NanoCav, LLC, United States
  5. NantBio, Inc, and ImmunityBio, Inc, United States
  6. NantOmics, LLC, United States
  7. California NanoSystems Institute, University of California, Los Angeles, United States
  8. Department of Bioengineering, University of California, Los Angeles, United States
  9. Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research University of California, Los Angeles, United States
  10. Department of Pediatrics, David Geffen School of Medicine, University of California, Los Angeles, United States
  11. Jonsson Comprehensive Cancer Center, David Geffen School of Medicine, University of California, Los Angeles, United States
https://doi.org/10.7554/eLife.63102
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Cite this article
  1. Alexander J Sercel
  2. Alexander N Patananan
  3. Tianxing Man
  4. Ting-Hsiang Wu
  5. Amy K Yu
  6. Garret W Guyot
  7. Shahrooz Rabizadeh
  8. Kayvan R Niazi
  9. Pei-Yu Chiou
  10. Michael A Teitell
(2021)
Stable transplantation of human mitochondrial DNA by high-throughput, pressurized isolated mitochondrial delivery
eLife 10:e63102.
https://doi.org/10.7554/eLife.63102
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Abstract

Generating mammalian cells with specific mitochondrial DNA (mtDNA)–nuclear DNA (nDNA) combinations is desirable but difficult to achieve and would be enabling for studies of mitochondrial-nuclear communication and coordination in controlling cell fates and functions. We developed ‘MitoPunch’, a pressure-driven mitochondrial transfer device, to deliver isolated mitochondria into numerous target mammalian cells simultaneously. MitoPunch and MitoCeption, a previously described 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.

eLife digest

Mitochondria are specialized structures within cells that generate vital energy and biological building blocks. Mitochondria have a double membrane and contain many copies of their own circular DNA (mitochondrial DNA), which include the blueprints to create just thirteen essential mitochondrial proteins.

Like all genetic material, mitochondrial DNA can become damaged or mutated, and these changes can be passed on to offspring. Some of these alterations are linked to severe and debilitating diseases. Both the double membrane of the mitochondria and their high number of DNA copies make treating such diseases difficult. A successful therapy must be capable of correcting almost every copy of mitochondrial DNA. However, the multiple copies of mitochondrial DNA create a problem for genetic research as current techniques are unable to reliably introduce particular mitochondrial mutations to all types of human cells to investigate how they may alter cell function.

Sercel, Patananan et al. have developed a method to deliver new mitochondria into thousands of cells at the same time. This technique, called MitoPunch, uses a pressure-driven device to propel mitochondria taken from donor cells into recipient cells without mitochondrial DNA to reestablish their function. Using human cancer cells and healthy skin cells that lack mitochondrial DNA, Sercel, Patananan et al. showed that cells that received mitochondria retained the new mitochondrial DNA. The technique uses readily accessible parts, meaning it can be performed quickly and inexpensively in any laboratory. It further only requires a small amount of donor starting material, meaning that even precious samples with limited material could be used as mitochondrial donors.

This new technique has several important potential applications for mitochondrial DNA research. It could be used in the lab to create large numbers of cell lineswith known mutations in the mitochondrial DNA to establish new systems that test drugs or probe the interaction between mitochondrial and nuclear DNA. It could be used to study a broad spectrum of biological questions since mitochondrial function is essential for several processes required for life. Critically, it could also be used as a starting point to develop next-generation therapies capable of treating inherited mitochondrial genetic diseases in severely affected patients.

Introduction

Mitochondrial DNA (mtDNA) and nuclear DNA (nDNA) genome coordination regulates metabolism, epigenome modifications, and other processes vital for mammalian cell survival and activity (Patananan et al., 2018; Ryan and Hoogenraad, 2007; Singh et al., 2017). Together, these genomes encode >1100 mitochondrial proteins, with only 13 essential electron transport chain (ETC) proteins encoded within the mtDNA (Calvo and Mootha, 2010). The mitochondrial proteins encoded in the mtDNA and the nDNA must be compatible to support mitochondrial ETC activity. Mutations in mtDNA can impair the ETC by altering nDNA co-evolved ETC complex protein interactions, causing defective cellular respiration and debilitating diseases (Greaves et al., 2012). Furthermore, the coordination of these two genomes to transcribe, translate, and potentially modify appropriate levels of their respective gene products to maintain energetic and metabolic homeostasis is essential to the proper functioning of the ETC (Wolff et al., 2014). As a result, methods that enable pairing of specific mtDNA and nDNA genotypes in tractable systems are key to understanding the basic biology of mitonuclear interactions and their implications for health and disease.

Our current inability to edit mtDNA sequences is a roadblock for many studies and potential applications. For example, endonucleases targeted to the mitochondrion inefficiently eliminate and cannot alter mtDNA sequences (Bacman et al., 2018). An exciting new bacterial cytidine deaminase toxin generates a limited repertoire of point mutations in the mtDNA; however, its efficiency remains low and it is unable to knock-in new gene sequences (Mok et al., 2020). Mitochondrial transfer between cells in vitro and in vivo provides a potential path forward for transplanting existing mtDNA sequences; however, the mechanisms controlling such transfers remain unknown (Dong et al., 2017; Torralba et al., 2016). Isolated mitochondrial transfer has been used to deliver mitochondria to a range of recipient cell types in vitro and even in vivo (Caicedo et al., 2015; Emani et al., 2017; Kitani et al., 2014); however, most studies using these methods observe only short-term changes to cell or organ performance and function. A small number of these studies have coincubated mitochondria with recipient cells and observed permanent retention of the exogenous mtDNA in mtDNA-deficient (so-called ‘ρ0’) cells using large doses of mitochondria or antibiotic selection schemes (Clark and Shay, 1982; Patel et al., 2017), although these approaches may not be possible when mitochondrial donor material is limited or does not possess a suitable selection marker.

Methods that deliver mitochondria directly into ρ0 cells can increase stable mitochondrial transfer efficiency and employ a wider range of mitochondrial donor sources. Such methods include membrane disruption (King and Attardi, 1988; Wu et al., 2016) or fusion with enucleated cytoplasts (Wilkins et al., 2014). However, these methods are typically laborious, low-throughput, or depend on cancerous, immortal recipient cells lacking physiologic mitochondrial activity. An interesting recent study did report one desired mtDNA–nDNA clone and 11 false-positive clones using cybrid fusion with replication-limited cells, an achievement hampered by a low generation rate with unknown reproducibility or generalizability (Wong et al., 2017).

There exist clinically relevant methods to replace the mtDNA of human cells, such as somatic cell nuclear transfer and pronuclear transfer that involve delivering nuclear genetic material from patients with mtDNA diseases into enucleated oocytes with non-mutant mtDNA genotypes (Hyslop et al., 2016; Tachibana et al., 2013). These methods hold potential for replacing deleterious mtDNA for the unborn, but they are technically challenging, low-throughput, dependent on high-quality patient samples, and prone to contamination by mutant mtDNA from the affected nuclear source material (Kang et al., 2016). Higher-throughput techniques that exchange non-native for resident mtDNAs in non-immortal somatic cells in tissue culture could enable studies of mtDNA–nDNA interactions and replace deleterious mtDNAs within cells with therapeutic potential (Patananan et al., 2016). Thus, a higher throughput, reproducible, and versatile mtDNA transfer approach to generate multiple desired ‘stable isolated mitochondrial recipient’ (SIMR) clones in replication-limited cells remains essential for statistically valid studies and potential translation of mitochondrial transplantation.

Results

MitoPunch mechanism uses fluid pressure to disrupt the plasma membrane

We developed ‘MitoPunch’ as a simple, high-throughput mitochondrial transfer device consisting of a lower polydimethylsiloxane (PDMS) reservoir loaded with a suspension of isolated mitochondria, covered by a polyethylene terephthalate (PET) filter seeded with ~1 × 105 adherent cells (Figure 1A, Figure 1—figure supplement 1). MitoPunch uses a solenoid-activated plunger to transfer isolated mitochondria in a holding chamber by force into the cytosol of mammalian cells. Upon actuation, a mechanical plunger deforms the PDMS from below, which, as calculated by numerical simulation, generates pressure up to 28 kPa inside the PDMS chamber, propelling the suspension through numerous 3 µm pores in the PET filter. This pressure cuts the plasma membrane of recipient cells sitting atop the pores and delivers mitochondria into the cytoplasm of the cut cells (Figure 1B). To assess performance, we compared MitoPunch to mitochondrial coincubation (Kitani et al., 2014) and to MitoCeption (Caicedo et al., 2015), a method that uses centripetal force generated in a centrifuge to localize mitochondria to recipient mammalian cells (Figure 1C). In MitoCeption, a 1500 × g centripetal force draws isolated mitochondria to a recipient cell monolayer. We calculate that the suspended mitochondria exert a pressure of ~1.6 Pa on recipient cell membranes (Figure 1D) (see Materials and methods).

Figure 1 with 1 supplement see all
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Pressure simulations of mitochondrial transfer tools.

(A) Schematic of MitoPunch apparatus. Recipient cells (1 × 105) are seeded on a porous polyester (PET) membrane ~24 hr before delivery. A freshly isolated suspension of mitochondria in 1× Dulbecco's Phosphate Buffered Saline (DPBS) with calcium and magnesium, pH 7.4, is loaded into the polydimethylsiloxane (PDMS) chamber and the filter insert is sealed over the PDMS before activation of the mechanical plunger to pressurize the apparatus and deliver the mitochondrial suspension into recipient cells. (B) Numerical simulation showing the pressure inside the PDMS chamber reaching 28 kPa with piston activation. COMSOL file used to model MitoPunch pressure is available in Figure 1—source data 1. (C) Schematic of MitoCeption technique. Recipient cells (1 × 105) are seeded on wells of a 6-well dish ~24 hr before delivery. A freshly isolated suspension of mitochondria in 1× DPBS with calcium and magnesium, pH 7.4, is pipetted into the cell medium before the plate is centrifuged at 1500 × g for 15 min at 4°C. The plate is incubated in a 37°C incubator for 2 hr before being centrifuged again at 1500 × g for 15 min at 4°C. (D) MitoCeption pressure model and calculated pressure exerted by isolated mitochondria on recipient cells during delivery.

Figure 1—source data 1

Numerical simulation of MitoPunch pressure generation during mitochondrial delivery.

https://cdn.elifesciences.org/articles/63102/elife-63102-fig1-data1-v2.zip

Mitochondrial delivery into transformed and primary cells

We isolated and delivered dsRed-labeled mitochondria from ~1.5 × 107 HEK293T cells (Miyata et al., 2014) into ~1 × 105 143BTK– ρ0 osteosarcoma cells and replication-limited BJ ρ0 foreskin fibroblasts in technical triplicate and measured the fraction of recipient cells positive for dsRed fluorescence by ImageStreamx MarkII imaging flow cytometry (Figure 2A). We define technical replication as independently performed mitochondrial deliveries using the same isolated mitochondrial preparation into recipient cells of the same passage. For 143BTK– ρ0 cells at ~2 hr post-delivery, imaging flow cytometry showed that MitoPunch yielded the lowest fraction of dsRed-positive cells compared to coincubation or MitoCeption. Similarly, for BJ ρ0 recipient cells, MitoPunch yielded the lowest fraction of dsRed-positive cells compared to coincubation or MitoCeption, although at lower levels relative to 143BTK– ρ0 recipients. This measurement assesses colocalization of mitochondria with recipient cells, and not necessarily the occurrence or mechanism of internalization of delivered mitochondria. These data suggest that the method of delivery and target cell type affect the efficiency of initiating mitochondria–recipient cell interactions.

Figure 2 with 1 supplement see all
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MitoPunch delivers isolated mitochondria to recipient cells.

(A) Quantification of flow cytometry results measuring the association of dsRed mitochondria with 143BTK– ρ0 and BJ ρ0 single recipient cells following mitochondrial transfer. (B) Mean and median dsRed spot count quantification of ImageStream data. (C) Sequential Z-stacks of confocal microscopy of 143BTK– ρ0 cells delivered isolated HEK293T-derived dsRed mitochondria by coincubation, MitoPunch, and MitoCeption and fixed 15 min following transfer. Arrows indicate representative mitochondria interacting with recipient cells. Transferred dsRed mitochondria are labeled in red. Plasma membranes are labeled in green, stained with CellMask Green plasma membrane stain in coincubation and MitoCeption and with wheat germ agglutinin plasma membrane stain in MitoPunch. Scale bars indicate 15 µm. (D) Quantification of flow cytometry measurements of fluorescence in 143BTK– ρ0 and BJ ρ0 single cells following propidium iodide transfer by coincubation, MitoPunch, and MitoCeption. Error bars represent SD of three technical replicates in all figures.

We quantified the number of discreet dsRed-spots in each cell ~2 hr following delivery from this data (George et al., 2004; Figure 2B and Figure 2—figure supplement 1). ImageStream spot count analysis of 143BTK– ρ0 recipient cells showed MitoPunch delivered a lower mean and median number of dsRed spots per cell than coincubation or MitoCeption. MitoPunch transfers into BJ ρ0 recipient cells yielded fewer mean spots/cell compared to coincubation and MitoCeption with an equivalent median number of spots/cell for MitoPunch and MitoCeption and slightly more for coincubation. Next, we used confocal microscopy to observe dsRed mitochondrial fluorescence in 143BTK– ρ0 recipients fixed 15 min post-transfer, which we chose for its robust mitochondrial acquisition (Figure 2C). We visualized mitochondrial localization with confocal microscopy by detecting dsRed protein from the donor mitochondria, shown in red, and labeling the recipient cell plasma membranes with either CellMask Green (coincubation and MitoCeption) or wheat germ agglutinin (MitoPunch), shown in green. Following MitoPunch, mitochondrial dsRed appeared to localize to pores in the filter insert and within the cytoplasm of cells, whereas coincubation and MitoCeption uniformly coated recipient cells with mitochondria, with greater mitochondrial association with recipient cells following MitoCeption. While all three methods initiate physical interactions between mitochondria and recipient cells, MitoPunch delivers mitochondria to the basal membranes of recipient cells at regions associated with the PET membrane pores, compared to a diffuse membrane association pattern seen with coincubation and MitoCeption.

To investigate the capacity of these methods to disrupt recipient cell plasma membranes, we delivered the membrane impermeant dye propidium iodide (PI) by coincubation, MitoPunch, and MitoCeption to measure membrane disruption from delivery and quantified uptake by flow cytometry (Figure 2D; Novickij et al., 2017). Delivery into 143BTK– ρ0 cells by MitoPunch and MitoCeption resulted in similar percentages of PI-positive recipient cells, and both were greater than coincubation. Interestingly, BJ ρ0 cells showed comparable fractions of PI-positive cells to the 143BTK– ρ0 after coincubation and MitoCeption. However, MitoPunch yielded an approximately fivefold increase in the PI-positive fraction compared to all other conditions. These data show that MitoPunch and MitoCeption disrupt the plasma membranes of recipient cells for potential mitochondrial transfer, and the degree of disruption is cell type and delivery method dependent.

Stable retention of transplanted mtDNA

After verifying mitochondrial interaction with recipient cells by coincubation, MitoPunch, and MitoCeption, we next determined whether these methods result in permanent retention of exogenous mtDNA to generate SIMR cells. ρ0 cells cannot synthesize pyrimidines and therefore cannot proliferate or survive without supplemented uridine because of ETC impairment, so we used nucleotide-free medium prepared with dialyzed fetal bovine serum (SIMR selection medium) to select for SIMR cells with transplanted mtDNA and restored ETC activity (Grégoire et al., 1984; Figure 3A and B). BJ ρ0 cells survive longer under this selection scheme compared to the 143BTK– ρ0 (data not shown), so we included an additional selection phase by culturing these cells in nucleotide-free, glucose-free, galactose supplemented medium (galactose selection medium) (Robinson et al., 1992). We isolated and transferred HEK293T dsRed mitochondria into 143BTK– ρ0 and BJ ρ0 cells by coincubation, MitoPunch, and MitoCeption, performed SIMR selection in cell-type appropriate medium for 7 days, and quantified the number of viable clones by crystal violet staining (Figure 3C). Coincubation did not generate SIMR clones in 143BTK– ρ0 cells, in contrast to MitoPunch and MitoCeption, which each generated dozens of clones. BJ ρ0 cells with delivered HEK293T mitochondria by coincubation or MitoCeption did not form SIMR clones. MitoPunch generated numerous SIMR clones in both cell types, although fewer BJ ρ0 SIMR clones than in 143BTK– ρ0 cells, whereas MitoCeption only generated clones in 143BTK– ρ0 cells and was unable to form stable clones in replication-limited BJ cells. To assess the risk of mitochondrial donor cells surviving disruption during mitochondrial isolation and generating false positive SIMR clones, we performed three independent mitochondrial isolations, plated an aliquot from each isolation representing mitochondria isolated from ~1.5 × 107 HEK293T dsRed cells on 10 cm dishes, and carried these plates through the 10-day selection with SIMR selection medium before crystal violet staining for visual assessment (Figure 3—figure supplement 1). We observed no cell growth on any of the three plates, indicating a minimal incidence of donor cell survival through the mitochondrial isolation protocol.

Figure 3 with 6 supplements see all
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Stable retention of transplanted mitochondrial DNA (mtDNA) into transformed and replication-limited cells.

(A) Workflow for stable isolated mitochondrial recipient (SIMR) cell generation by mitochondrial transfer into ρ0 cells. (B) Representative fixed and crystal violet stained 10 cm plate image following MitoPunch and SIMR cell selection used for SIMR clone generation quantification. (C) Quantification of crystal violet stained 143BTK– ρ0 and BJ ρ0 SIMR clones. Error bars represent SD of three technical replicates. (D) Quantification of crystal violet stained 143BTK– ρ0 and BJ ρ0 SIMR clones formed by MitoPunch actuated with indicated voltages after uridine-free selection. Error bars represent SD of three technical replicates with the exception of BJ ρ0 5 V transfer, which shows two replicates. (E) Quantification of crystal violet stained 143BTK– ρ0 and BJ ρ0 SIMR clones formed by MitoCeption with indicated centripetal forces after uridine-free selection. Error bars represent SD of three technical replicates.

We next investigated whether differences in SIMR clone generation between 143BTK– ρ0 and BJ ρ0 cells were driven by sensitivity to differences in delivery pressure. We developed a MitoPunch device with adjustable plunger acceleration modulated by changing the circuit voltage (ImmunityBio). We generated independent voltage titration curves in 1 V increments (0 V – 5 V) for each cell type in technical triplicate at each voltage and used the same mitochondrial preparation for all samples for each cell type. All prior experiments in this study are controlled by delivering DPBS with calcium and magnesium to recipient cells by MitoPunch, but here we included a 0 V condition in which the seeded filter insert was positioned atop the PDMS reservoir and pressed against an aliquot of isolated mitochondrial suspension similar to deliveries with force, but without actuating the piston. We achieved maximum 143BTK– ρ0 SIMR clone generation with this tunable MitoPunch at 1 V, with a sharp reduction to background with increasing voltage (Figure 3D). The BJ ρ0 recipient also showed maximal SIMR generation at 1 V, with a shallow decline in SIMR generation efficiency to 5 V. Surprisingly, the 0 V condition consistently yielded a few SIMR clones in the 143 BTK– ρ0 recipients and inconsistently in the BJ ρ0 recipients. This result suggests that the pressure generated by sealing the filter insert against the PDMS reservoir is sufficient to generate SIMR clones at a low frequency. For all forthcoming MitoPunch trials we use the variable voltage MitoPunch device set to 1 V.

We performed a similar force titration with MitoCeption by varying the maximum centripetal force, using a common mitochondrial preparation for all samples of both cell types. In 143BTK– ρ0 cells, we observed maximum clone generation at 1000 × g and 1500 × g, and we did not generate BJ ρ0 SIMR clones greater than the 0 × g background at any acceleration tested (Figure 3E). This background, present in both 143BTK– ρ0 and BJ ρ0 conditions at 0 × g, is likely from rare un-lysed donor cells from mitochondrial preparations directly pipetted into the culture medium of recipient cells during MitoCeption. We have infrequently observed imperfect donor cell lysis , usually in larger mitochondrial preparations, that results in rare, persistant dsRed fluorescent colonies as observed by fluorescence microscopy. True SIMR clones cannot produce the dsRed protein from donor mitochondria and lose fluorescence with time over selection, while these persistent dsRed colonies maintain their fluorescence over the same period (data not shown). Despite this occasional low-level contaminating donor cell background, MitoCeption yielded a strong dose-dependent response in SIMR clone generation from 143BTK– ρ0 recipients above the background. Additionally, MitoPunch deliveries into B16 ρ0 mouse melanoma cells (Tan et al., 2015) yielded maximal SIMR generation at a different voltage than in the human cell lines tested, showing that optimal mitochondrial delivery pressure may be cell type dependent (Figure 3—figure supplement 2). These data suggest that MitoPunch is uniquely able to generate SIMR clones in replication-limited fibroblasts and SIMR generation efficiency depends on delivery pressure.

We next quantified the reproducibility of our mitochondrial preparation technique and the MitoPunch procedure by performing triplicate MitoPunch transfers using three independent mitochondrial preparations from equal numbers of HEK293T dsRed biological replicate populations (Figure 3—figure supplements 3 and 4). We define biological replication here as mitochondrial preparations derived from independently cultured populations of mitochondrial donor cells. Mitochondrial preparations 1, 2, and 3 (same as those pictured in Figure 3—figure supplement 1) generated consistent protein concentrations, and each preparation yielded dozens of SIMR clones in all three technical replicate MitoPunch deliveries with the exception of Prep 3, which resulted in two lower efficiency replicates. We quantified the number of SIMR clones generated per microgram of mitochondrial mass loaded into the MitoPunch apparatus and observed a similar trend. These results showed that our mitochondrial isolation technique produced consistent levels of isolated mitochondrial mass and that the MitoPunch technique yielded high numbers of SIMR clones.

To enable desirable mtDNA–nDNA clone generation using limited starting material, such as mitochondria from rare cell subpopulations, we determined the minimal mass of mitochondrial isolate required to generate SIMR clones. We performed coincubation, MitoPunch, and MitoCeption transfers into ~1 × 105 143BTK– ρ0 recipient cells using decreasing concentrations of dsRed mitochondria isolated from HEK293T cells and plated half of the recipient cell population on 10 cm plates. We observed a similar dose-dependent relationship between mitochondrial mass delivered and SIMR clones observed for MitoPunch and MitoCeption across 0.16 µg, 1.6 µg, and 16 µg total mitochondrial protein suspended in 120 µL of 1× DPBS, pH 7.4 transfer buffer (Figure 3—figure supplement 5). These results showed that although MitoPunch and MitoCeption generate SIMR clones from transformed recipient cells with similar efficiency per microgram of mitochondrial isolate delivered, the differences inherent to the two protocols rendered direct comparisons of their relative efficiencies less meaningful.

Moving the seeded PET filter from a 12-well dish to the MitoPunch apparatus often resulted in excess medium being carried to the PDMS reservoir. Combined with the small volume of mitochondrial preparation delivered to the recipient cells, we observed that MitoPunch resulted in diluted residual mitochondrial isolate left in the reservoir post-transfer. In the interest of conserving mitochondrial material, we tested whether a used 120 µL aliquot of isolated mitochondria can be applied to repeated MitoPunch transfers to generate SIMR clones (Figure 3—figure supplement 6). We performed 11 sequential deliveries into 143BTK– ρ0 cells using one aliquot of mitochondrial isolate and found maximal SIMR clone generation from the first and second deliveries, after which we observe a sharp reduction in SIMR cell formation and inconsistent SIMR generation rate up to the 11th transfer. These data showed that multiple MitoPunch transfers can be performed using a single aliquot of mitochondrial suspension when material is limited.

SIMR cells rescue ρ0 mitochondrial respiration and network morphology

Finally, we measured mitochondrial function in SIMR cells by quantifying the rate of oxygen consumption and assessing mitochondrial morphology. We isolated three independent 143BTK– ρ0 SIMR clones generated by MitoPunch or MitoCeption transfer of isolated HEK293T mitochondria and measured each clone’s oxygen consumption rate (OCR) using a Seahorse Extracellular Flux Analyzer mitochondrial stress test (Figure 4A, Figure 4—figure supplement 1). To determine whether SIMR clone respiration remained stable through time, we grew the clones through two freeze/thaw cycles in uridine supplemented medium and measured cellular respiration. We found that one MitoCeption clone lost its respiratory capacity and one MitoPunch clone was not viable after freezing and thawing (data not shown). In the remaining clones, basal and maximal respiration, spare respiratory capacity, and ATP generation remained stable throughout both freeze-thaw cycles. We have performed numerous similar experiments using a range of recipients and mitochondrial donors and observed successful clone viability after freeze-thaw (data not shown).

Figure 4 with 2 supplements see all
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Mitochondrial DNA (mtDNA) transplantation rescues ρ0 mitochondrial phenotypes.

(A) Oxygen consumption rate (OCR) quantification of basal and maximal respiration, spare respiratory capacity, and ATP generation from two independent 143BTK– ρ0 + HEK293T stable isolated mitochondrial recipient (SIMR) clones generated by MitoPunch and MitoCeption. Cross-hatched data indicate clones that were frozen and thawed twice each. Error bars represent SD of four technical replicates for fresh SIMR cell measurements and five for thawed SIMR cell measurements. (B) Confocal microscopy of representative 143BTK– ρ0 + HEK293T SIMR clones compared to 143BTK– parental, HEK293T dsRed mitochondrial donor, and 143BTK– ρ0 controls. Mitochondria were stained with anti-TOM20 antibody and labeled red, double-stranded DNA was stained with anti-dsDNA antibody and labeled green, and cell nuclei were stained with NucBlue (Hoechst 33342) and labeled blue. Scale bars indicate 15 µm.

We then immunostained the freeze-thawed SIMR clones with anti-TOM20 and anti-double-stranded DNA (dsDNA) antibodies to detect mitochondria and mtDNA content, respectively, by confocal microscopy (Figure 4B and Figure 4—figure supplement 2). The MitoCeption clone that lost respiratory capacity showed a fragmented mitochondrial network with no detectable mtDNA (Figure 4—figure supplement 2), whereas the other SIMR clones generated by MitoPunch and MitoCeption contained mtDNA with filamentous mitochondrial network morphologies. These data show that the majority of 143BTK– ρ0 SIMR clones generated by either MitoPunch or MitoCeption have retained mtDNA, restored respiratory profiles, and filamentous mitochondrial network morphologies.

Discussion

Stability of the mitochondrial genome is essential for studying the long-term effects of mtDNA–nDNA interactions and for potential therapeutic applications of mitochondrial transfer. MitoPunch generates up to hundreds of SIMR clones in both transformed and Hayflick-limited recipient cells by exerting a pressure sufficient to perforate mammalian cell membranes in regions small enough to be repaired within minutes, which sustains cell viability and resumed cell growth and proliferation (Boye et al., 2017). We have generated SIMR clones by MitoPunch with mitochondria isolated by a commercially available kit, as performed here, as well as by using standard mitochondrial isolation buffers. Additionally, we achieved similar results by disrupting mitochondrial donor cells using Dounce homogenization (data not shown) but found the commercially available kit with syringe disruption is advantageous due to its ease of use, reproducibility, and a reduced number of steps to isolate mitochondria. We generated dozens of SIMR clones by MitoPunch and MitoCeption using these mitochondrial isolation methods and anticipate that other mitochondrial preparation techniques will also yield SIMR clones.

Interestingly, we do not observe SIMR clone generation by coincubation in our study. Few reports show limited stable clone formation by coincubation techniques, but these studies used up to 100-fold higher levels of exogenous mitochondria in coincubation experiments than required for MitoPunch or MitoCeption in our hands, or antibiotic selection schemes to achieve stable mitochondrial transfer (Clark and Shay, 1982; Patel et al., 2017). High levels of mitochondrial protein are easily isolated from fast-growing immortalized cell lines but may not be available when using human donor-derived or other limiting starting material. Additionally, mitochondrial donor cells of interest nearly exclusively lack antibiotic selection markers, making such selection schemes unfeasible. Particularly in those cases, the greatly enhanced SIMR generation capacity of MitoPunch and MitoCeption is strongly enabling for generating desired mtDNA–nDNA combinations.

The distinct mechanisms and procedures of MitoPunch and MitoCeption make direct comparisons of their relative efficiencies challenging. Despite this, our results demonstrate that both techniques generate SIMR clones from ρ0 transformed cells in a mitochondrial dose-dependent fashion and can be readily adopted by laboratories studying mtDNA–nDNA interactions. Strikingly, in the cell types we have tested, we find that only MitoPunch generates SIMR clones from ρ0 primary, non-immortal cells. Studies in our laboratory suggest that the transcriptome and metabolome of replication-limited SIMR clones differ significantly from un-manipulated control clones but can be recovered and reset to un-manipulated control levels by cellular reprogramming to induced pluripotent stem cells and subsequent differentiation (Patananan et al., 2020). These results indicate that SIMR clone generation in replication-limited, reprogrammable cells is crucial for studies of mtDNA–nDNA interactions involving mitochondrial transplantation into ρ0 cells, and that MitoPunch is uniquely capable of efficiently generating enough clones for statistically valid studies in such work. We have circumvented the need for ρ0 recipient cells by using the MitoPunch technology to completely replace mutant mtDNA in mouse cells without mtDNA depletion. This was done by delivering mitochondria containing mtDNA with a chloramphenicol resistant point mutation and selecting for SIMR clones containing only rescue mtDNA using antibiotic supplemented nucleotide-free medium (Dawson et al., 2020). However, this workflow is dependent upon using antibiotic resistant mitochondrial donor cells and is not applicable to investigating the full spectrum of mtDNA sequences required for robust studies of mtDNA–nDNA interactions. Future work with MitoPunch and other isolated mitochondrial transfer modalities will be improved by developing techniques to avoid fully depleting the mtDNA of recipient cells of interest before generating SIMR clones for downstream analysis and applications.

Materials and methods

Key resources table
Reagent type
(species) or resource		DesignationSource or referenceIdentifiersAdditional information
Cell line (Homo sapiens)143 BTK– ρ0 osteosarcomaPatananan et al., 2020,
ATCC		Cat. #CRL-8303; RRID:CVCL_9W36
Cell line (Homo sapiens)143 BTK– osteosarcomaATCCCat. #CRL-8303; RRID:CVCL_9W36
Cell line (Homo sapiens)BJ ρ0 foreskin fibroblast (male)Patananan et al., 2020
ATCC		Cat. #CRL-2522; RRID:CVCL_3653
Cell line (Homo sapiens)HEK293T dsRedMiyata et al., 2014A gift from the laboratory of Dr. Carla Koehler
Cell line (M. musculus)B16 ρ0 melanomaDong et al., 2017A gift from the laboratory of Dr. Michael Berridge
Cell line (M. musculus)L929 fibroblastsATCCCat. #CCLl-1
AntibodyAnti-TOMM20 (Rabbit monoclonal)AbcamCat. #ab78547
RRID:AB_2043078		IF(1:1000)
AntibodyAnti-dsDNA (Mouse monoclonal)AbcamCat. #ab27156
RRID:AB_470907		IF(1:1000)
AntibodyAnti-rabbit IgG (Donkey polyclonal)Thermo Fisher ScientificCat. #A-31573
RRID:AB_2536183		IF(1:100)
AntibodyAnti-mouse IgG (Donkey polyclonal)Thermo Fisher ScientificCat. #A-21202
RRID:AB_141607		IF(1:100)
Commercial assay or kitQproteome Mitochondria Isolation kitQiagenCat. #37612
Commercial assay or kitBCA protein assayThermo FisherCat. #23225
Chemical compound, drugPropidium iodideThermo Fisher ScientificCat. #P1304MP
Chemical compound, drugAccutaseThermo Fisher ScientificCat. #A1110501
Chemical compound, drug16% paraformaldehydeThermo Fisher ScientificCat. #28906
Chemical compound, drugTriton-X 100SigmaCat. #X100
Chemical compound, drugProLong Gold Antifade Mountant with DAPIInvitrogenCat. #P3691
Chemical compound, drugProLong Glass Antifade Mountant with NucBlue StainThermo Fisher ScientificCat. #P36985
Chemical compound, drugUridineThermo Fisher ScientificCat. #AC140770250
Chemical compound, drugGalactoseSigma-AldrichCat. #G5388-100G
Chemical compound, drugCellMask Green PMMolecular ProbesCat. #C37608
Chemical compound, drugAlexa Fluor 488 conjugated Wheat Germ AgglutininInvitrogenCat. #W11261
Chemical compound, drugCrystal violetThermo Fisher ScientificCat. #C581-25
Software, algorithmWave 2.6.2AgilentRRID:SCR_014526
Software, algorithmFlowJo 10.6.2BD BiosciencesRRID:SCR_008520
Software, algorithmIDEAS 6.2Luminex
Software, algorithmMultiphysics 5.3COMSOLRRID:SCR_014767
Software, algorithmImaris Viewer 9.5.1Oxford InstrumentsRRID:SCR_007370
Software, algorithmImaris File Converter 9.5.1Oxford InstrumentsRRID:SCR_007370
Software, algorithmPrism v.8GraphpadRRID:SCR_002798
Software, algorithmLAS X Lite 3.7.1.21655Leica
Software, algorithmFIJISchindelin et al., 2012
OtherDialyzed FBSLife TechnologiesCat#26400–044
Other12–well 3.0 µm Transparent PET MembraneCorningCat#353181
OtherGlass coverslipsZeissCat#474030–9000
OtherV3 96-well plateAgilentCat#101085–004
OtherVariable voltage MitoPunch apparatusImmunityBioInquiries regarding this device can be made to the corresponding author

Cell culture conditions

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Human ρ0 cells were grown in DMEM (Fisher Scientific, Waltham, MA, Cat. # MT10013CM) supplemented with 10% FBS, non-essential amino acids (Gibco, Waltham, MA, Cat. #11140–050), GlutaMax (Thermo Fisher Scientific, Waltham, MA, Cat. # 35050–061), penicillin and streptomycin (VWR, Radnor, PA, Cat. # 45000–652), and 50 mg/L uridine (Thermo Fisher Scientific, Cat. # AC140770250). All other human cell lines were grown in DMEM (Fisher Scientific, Cat. # MT10013CM) supplemented with 10% FBS, non-essential amino acids, GlutaMax, and penicillin and streptomycin. B16 ρ0 cells were grown in RPMI (Thermo Fisher Scientific, Cat. # MT-10–040 CM) supplemented with 10% FBS, non-essential amino acids, GlutaMax, penicillin and streptomycin, pyruvate (Corning, Corning, NY, Cat. # 25000 CI), and 50 mg/L uridine. L929 cells were grown in RPMI supplemented with 10% FBS, non-essential amino acids, GlutaMax, penicillin and streptomycin, and pyruvate. All mammalian cells were cultured in a humidified incubator maintained at 37°C and 5% CO2. The following cells were used in this study: HEK293T dsRed (female), 143BTK– (female), 143BTK– ρ0 (female), BJ ρ0 (male), B16 (male), and L929 (male). We have not formally identified these cell lines; however, we have sequenced their mitochondrial and nuclear DNA for polymorphisms and find unique sequences which we use for genotyping our cultures (unpublished data). BJ ρ0 cells were used as mitochondrial recipients within three passages of thaw for all mitochondrial transfer experiments in this work to avoid the onset of senescence. All lines were routinely tested for mycoplasma with negative results.

Mitochondrial isolation

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Mitochondria were isolated from ~1.5 × 107 mitochondrial donor cells per mitochondrial transfer using the Qproteome Mitochondrial Isolation Kit (Qiagen, Hilden, Germany, Cat. #37612) with slight alterations to the manufacturers protocol. Mitochondrial donor cells were harvested using a cell scraper (Fisher Scientific, Cat. # 08-100-241) and collected in 50 mL conical tubes at approximately 6 × 107 cells per tube (Thermo Scientific, Cat. #12-565-271). Cells were pelleted by centrifugation at 500 × g for 10 min at 4°C and washed with DPBS before pelleting again by centrifugation at 500 × g for 10 min at 4°C. Cells were resuspended at 1 × 107 cells/mL in ice-cold Lysis Buffer with Protease Inhibitor Solution and incubated for 10 min at 4°C in 2 mL tubes on an end-over-end shaker. Lysates were centrifuged at 1000 × g for 10 min at 4°C and supernatant was aspirated. Pellets were resuspended in 1.5 mL ice-cold Disruption Buffer with Protease Inhibitor Solution and mechanical disruption was accomplished by 10 passes through a 26 G blunt ended needle (VWR, Radnor, PA, Cat. # 89134–164) attached to a 3 mL syringe (VWR, Cat. # BD309657). The subsequent lysates were centrifuged at 1000 × g for 10 min at 4°C and the supernatants were transferred to new 2 mL tubes. The resultant supernatants were centrifuged again at 1000 × g for 10 min at 4°C to remove any remaining intact cells, and the supernatants were transferred to clean 1.5 mL tubes. These supernatants were centrifuged at 6000 × g for 10 min at 4°C and the supernatants were aspirated. The resulting mitochondrial pellets were resuspended in mitochondrial storage buffer and pelleted by centrifugation at 6000 × g for 20 min at 4°C. The isolated mitochondrial pellets were resuspended in 120 µL per transfer replicate 1× DPBS with calcium and magnesium (Thermo Fisher Scientific, Cat. # 14040133) immediately prior to mitochondrial transfer and kept on ice.

Mitochondrial coincubation

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~1 × 105 143BTK– ρ0 or BJ ρ0 cells were seeded into wells of 6-well dishes ~ 24 hr prior to delivery. Mitochondria isolated from ~1.5 × 107 HEK293T dsRed cells resuspended in 120 µL 1× DPBS with calcium and magnesium were pipetted into the culture medium of each well containing recipient cells and incubated at 37°C and 5% CO2 for 2 hr. Cells were then released from the dish using Accutase (Thermo Fisher Scientific, Cat. # A1110501) and seeded into 10 cm plates for SIMR cell selection or harvested for additional analyses.

MitoPunch apparatus construction

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A 5 V solenoid (Sparkfun, Boulder, CO, Cat. # ROB-11015) is screwed into a threaded plug (Thor Labs, Newton, NJ, Cat. # SM1PL) and inserted into a bottom plate (Thor Labs, Cat. # CP02T) (Figure 1—figure supplement 1). The solenoid is regulated by a Futurlec mini board (Futurlec, New York, NY, Cat. # MINIPOWER) and powered by a MEAN WELL power supply (MEAN WELL, New Taipei City, Taiwan, Cat. # RS-35–12). Optomechanical assembly rods (Thor Labs, Cat. # ER3) are inserted into the bottom plate. The middle and top plates (Thor Labs, Cat. # CP02) are threaded through the assembly rods. The middle plate is fitted with a retaining ring, which supports an aluminum washer (outer diameter, 25 mm; inner diameter, 10 mm). The middle plate is secured along the assembly rods using the included screws. The retaining ring is adjusted such that the top surface of the washer is at the same height as the piston surface in its retracted state. A flexible PDMS (10:1 ratio of Part A base: Part B curing agent) (Fisher Scientific, Cat. #NC9644388) reservoir consisting of a bottom layer (25 mm diameter, 0.67 mm height) bonded to an upper ring (outer diameter, 25 mm; inner diameter, 10 mm; height, 1.30 mm) is placed on top of the washer. This reservoir can contain up to ~120 µL of liquid. To perform MitoPunch delivery, a 3 µm membrane transwell insert (Corning, Cat. # 353181) seeded with 1 × 105 adherent cells is lowered through the top plate and rested atop one retaining ring. The insert is secured to the top plate by an additional retaining ring. This assembly is lowered until the base of the insert contacts the top surface of the PDMS reservoir and is secured in place with screws to form a tight seal. In addition, a variable voltage version of this device based on the same principles with identical delivery procedures as MitoPunch, but with tunable plunger acceleration achieved by varying actuator voltage, was engineered by ImmunityBio and is available upon request to the corresponding author. Optimal MitoPunch delivery voltage for individual cell lines is determined empirically by performing a voltage-response curve in technical triplicate across a range of voltages from 1 V to 5 V using the piston acceleration control software.

Seeding cells for MitoPunch mitochondrial transfer

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Filter inserts with 3 µm pores (Corning, Cat. # 353181) are placed in wells of a 12-well dish. 1.5 mL warm uridine supplemented medium is dispensed in the wells outside of the filter insert, and 1 × 105 adherent cells suspended in 0.5 mL warm uridine supplemented medium are seeded within the filter inserts and placed in a humidified incubator maintained at 37°C and 5% CO2 1 day prior to mitochondrial delivery.

MitoPunch mitochondrial transfer

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Following mitochondrial isolation, the MitoPunch apparatus is sterilized with 70% ethanol and entered into the biological safety cabinet and an autoclaved PDMS reservoir is placed in the device as indicated in Figure 1—figure supplement 1. The PDMS reservoir is washed 3× with 120 µL sterile DPBS with calcium and magnesium after being set in the MitoPunch apparatus. 120 µL mitochondrial suspension from ~1 × 107 donor cells in DPBS with calcium and magnesium is loaded into the PDMS reservoir. Mitochondrial transfer is performed by securing the seeded membrane to the PDMS reservoir and actuating the solenoid for 3 s. The mechanical plunger strikes the middle of the PDMS chamber, displacing the base layer by ~1.3 mm. This displacement pressurizes the mitochondrial suspension and propels it through the membrane and into the cells (Figure 1B). Once the solenoid has returned to its starting position, the insert is removed from the apparatus, placed back in the 12-well dish in its original medium, and incubated at 37°C and 5% CO2 for 2 hr. Cells were then released from the dish using Accutase and seeded into 10 cm plates for SIMR cell selection or harvested for additional analyses.

Collecting mitochondrial recipient cells following MitoPunch transfer

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Following MitoPunch mitochondrial transfer and 2 hr incubation, medium is aspirated from within the transwell filter with care taken not to disrupt the cells on the membrane, and then from outside and underneath the filter insert. The well and insert are washed 1× with DPBS (0.5 mL inside the insert and 1 mL outside the insert) with DPBS aspirated as before. Cells are released from the membrane by 5 min incubation at 37°C and 5% CO2 with Accutase (0.5 mL inside the insert and 1 mL outside the insert). Following incubation, the cells are suspended in the Accutase within the filter insert using a P1000 pipette, being careful not to puncture the PET membrane, and directly pipetted into 10 cm plates with 10 mL warm uridine supplemented medium.

MitoCeption

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As described previously (Caicedo et al., 2015), 1 × 105 recipient cells were seeded in each well of a 6-well dish and incubated at 37°C and 5% CO2 overnight. Mitochondrial isolate from ~1 × 107 donor cells suspended in 1× DPBS with calcium and magnesium was pipetted into the well and the plate was centrifuged at 1500 × g for 15 min at 4°C. Cells were removed from the centrifuge and incubated for 2 hr at 37°C and 5% CO2 before being centrifuged a second time at 1500 × g for 15 min at 4°C. Cells were then released from the dish using Accutase and seeded into 10 cm plates for SIMR cell selection or harvested for additional analyses.

The pressure generated by the MitoCeption method was estimated by calculating the force exerted per unit area of the cell membrane during centrifugation. The force induced by the centrifugation of a single mitochondrion on the cell membrane was equal to the centripetal force of the mitochondria under the acceleration of 1500 × g minus the buoyancy force,

Fcentrifugation=mmito- mwater*a

where mmito and mwater are the mass of mitochondria and water, and a is the acceleration rate of centrifugation. The equivalent pressure induced by mitochondria during centrifugation was approximated by

p=FcentrifugationS=mmito-mwater*aS= ρmito-ρwaterVaS≈ρmito-ρwater*a*d

where ρmito (1.1 g/cm3) and ρwater (1.0 g/cm3) are the density of mitochondria and water, V and S are the volume and cross-sectional area of mitochondria, and d is the thickness of a mitochondrion (~1 µm). Using values for the geometry and properties of a mitochondrion, the pressure induced by MitoCeption centrifugation was ~1.6 Pa.

Numerical simulation of MitoPunch internal pressure

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The finite element method (COMSOL Inc, Burlington, MA, Multiphysics 5.3) was used to simulate the pressure inside the MitoPunch PDMS chamber. We constructed the simulation geometry according to real device dimensions. Piston movement was applied as initial displacement in the y direction. Considering the incompressibility of the aqueous medium inside the PDMS chamber, the volume of the chamber was maintained constant while solving for the stress distribution of all the materials.

SIMR clone isolation

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Mitochondrial recipient and vehicle delivery control 143BTK– ρ0 cells were grown in complete medium supplemented with 50 mg/L uridine for 3 days following mitochondria or vehicle transfer. After 3 days, the medium was changed to SIMR selection medium (complete medium with 10% dialyzed FBS (Life Technologies, Carlsbad, CA, Cat. # 26400–044)) and medium was exchanged daily. After the vehicle delivery control sample died and clones emerged on mitochondrial transfer plates (~7 days SIMR selection medium), clones were isolated using cloning rings or plates were fixed and stained with crystal violet for counting.

Mitochondrial recipient and vehicle delivery control BJ ρ0 and B16 ρ0 cells were grown in complete media supplemented with 50 mg/L uridine for 3 days following mitochondria transfer. After 3 days, the medium was changed to SIMR selection medium and exchanged daily. On day 5 post-delivery, cells were shifted to galactose selection medium (glucose-free, galactose-containing medium [DMEM without glucose, Gibco, Cat. # 11966025] supplemented with 10% dialyzed FBS and 4.5 g/L galactose [Sigma-Aldrich, Cat. #G5388-100G]). After the vehicle delivery control sample died and clones emerged on mitochondrial transfer plates (~36 hr in galactose selection medium), clones were isolated using cloning rings or plates were fixed and stained with crystal violet for counting.

Crystal violet staining and clone counting

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Media was aspirated from 10 cm plates before fixation with 1 mL freshly diluted 4% paraformaldehyde in 1× DPBS for 15 min at RT. Fixative was removed and 1 mL 0.5% w/v crystal violet solution (Thermo Fisher Scientific, Cat. # C581-25) dissolved in 20% methanol in water was applied to each plate and incubated for 30 min at RT. Crystal violet was removed and plates were washed 2× with deionized water before drying overnight at RT. Dried plates were photographed and crystal violet stained clones were counted manually using FIJI (Schindelin et al., 2012).

Imaging flow cytometry

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Mitochondria were transferred to recipient cells, which were harvested and collected in 1.5 mL tubes. Samples were centrifuged 5 min at 1000 × g, supernatant was aspirated, and cells were washed 3× with 0.5 mL 1× DPBS, pH 7.4. The DPBS was aspirated and cells were fixed in 100 µL freshly diluted 4% paraformaldehyde (Thermo Fisher Scientific, Cat. # 28906) for 15 min on ice. Fixative was diluted with 1 mL of 1× DPBS, pH 7.4, and 5% FBS and centrifuged for 10 min at 500 × g. Supernatant was removed and cells resuspended in 1× DPBS, pH 7.4, with 5% FBS. Imaging flow cytometry was performed using an ImageStream MarkII platform and analyzed using the IDEAS 6.2 software package (Luminex, Austin, TX).

Confocal microscopy

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1 × 105 cells were plated in 6-well dishes with 2 mL of media on glass coverslips (Zeiss, Oberkochen, Germany, Cat. # 474030–9000) ~24 hr prior to sample preparation. Medium was aspirated and samples were fixed with 0.5 mL freshly diluted 4% paraformaldehyde in 1× DPBS, pH 7.4 pipetted onto samples and incubated for 15 min at RT. Paraformaldehyde was removed and samples were washed 3× with 5 min 1× DPBS incubations. Samples were then permeabilized by 10 min RT incubation in 0.1% Triton-X 100 (Sigma, St. Louis, MO, Cat. # X100). Permeabilized samples were washed 3× with 1× DPBS and then incubated for 1 hr at RT with 2% bovine serum albumin (BSA) dissolved in 1× DPBS blocking buffer. Blocking buffer was aspirated and cells incubated for 1 hr at RT with a 1:1000 dilution of primary antibodies in 2% BSA blocking buffer against dsDNA (Abcam, Cambridge, United Kingdom, Cat. # ab27156) and TOM20 protein (Abcam, Cat. # ab78547), and then washed 3× with 5 min 1× DPBS incubations. Cells were then incubated with secondary antibodies (Invitrogen, Cat. # A31573 and A21202) diluted 1:100 in 2% BSA blocking buffer protected from light for 1 hr at RT. After incubation with secondary antibodies, samples were washed 3× with 5 min 1× DPBS incubations and mounted on microscope slides.

To mount, samples were removed from the 6-well dish and rinsed by dipping in deionized water, dried with a Kimwipe, and mounted using ProLong Gold Antifade Mountant with DAPI (Invitrogen, Carlsbad, CA, Cat. # P3691) or ProLong Glass Antifade Mountant with NucBlue Stain (Thermo Fisher Scientific, Cat # P36985) on microscope slides (VWR, Cat. # 48311–601). Samples were dried at RT protected from light for 48 hr prior to confocal imaging with a Leica SP8 confocal microscope (Leica, Wetzlar, Germany) and later analyzed with either LAS X Lite 3.7.1.21655 (Leica) for two-dimensional image preparation or Imaris File Converter 9.5.1 (Oxford Instruments, Abingdon, United Kingdom) and Imaris Viewer 9.5.1 (Oxford Instruments) for Z-stack analysis.

To perform confocal imaging on cells immediately following mitochondrial transfer, 1 × 105 cells were plated in 6-well dishes with 2 mL of media on glass coverslips for coincubation and MitoCeption or seeded onto 12-well filter inserts as described above for MitoPunch ~24 hr prior to delivery. Immediately prior to mitochondrial transfer, coincubation and MitoCeption samples were stained with 1× CellMask Green PM (Molecular Probes, Eugene, OR, Cat. # C37608) diluted in warm medium for 10 min and washed twice in DPBS, and MitoPunch samples were stained with 5 µg/mL Alexa Fluor 488 conjugated Wheat Germ Agglutinin (Invitrogen, Cat. # W11261) diluted in warm media for 10 min and washed twice in DPBS. Following delivery, culture medium was removed and 1 mL freshly diluted 4% paraformaldehyde in 1× DPBS, pH 7.4, was pipetted onto samples and incubated for 15 min at RT. Paraformaldehyde was aspirated and samples were washed 3× with 1× DPBS, pH 7.4. Samples were further washed with DPBS 3× with 5 min RT incubation per wash. MitoPunch filters were removed from the plastic insert using an inverted P1000 pipette tip. Samples were mounted and imaged as described above.

OCR measurements

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OCR measurements were performed using a Seahorse XFe96 Extracellular Flux Analyzer (Agilent, Santa Clara, CA). 2 × 104 cells were seeded into each well of a V3 96-well plate (Agilent, Cat. # 101085–004) and cultured 24 hr before measuring OCR. The Agilent Seahorse mitochondrial stress test was used to quantify OCR for basal respiration and respiration following the sequential addition of the mitochondrial inhibitors oligomycin, carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP), and antimycin A. Data were analyzed using the Wave 2.6.2 software package (Agilent).

PI staining, delivery, and flow cytometry

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Cells (1 × 105) were plated for delivery and incubated overnight. Media was changed to FluorBrite DMEM media (ThermoFisher Scientific, Cat. # A1896701) with 3 µM PI (Thermo Fisher Scientific, Cat. # P1304MP) immediately before transfer. MitoCeption and coincubation were carried out as described above, and MitoPunch was performed with PI FluorBrite medium loaded into the PDMS reservoir and incubated for 15 min at 37°C and 5% CO2. All samples were washed with 1× DPBS and collected using Accutase. Samples were collected in flow cytometry tubes and centrifuged 5 min at 500 × g. Samples were washed with 1× DPBS with 5% FBS three times and analyzed on a BD Fortessa flow cytometer (BD Biosciences, San Jose, CA) and data were processed using FlowJo 10.6.2 (BD Biosciences).

Quantification and statistical analysis

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All information pertaining to experimental replication are found in the figure legends. Mitochondrial transfer experiments were performed in technical triplicate to enable calculation of standard deviation unless otherwise indicated, and oxygen consumption measurements were collected in technical quadruplicate or quintuplicate indicated in the legend of Figure 4. Investigators were blinded for SIMR colony counting analysis. All column heights represent the mean of technical triplicate results unless noted otherwise. All error bars in this manuscript represent standard deviation of three technical replicates unless otherwise specified in the figure legend.

Data availability

Figure 1-source data 1. Numerical simulation of MitoPunch pressure generation during mitochondrial delivery. Cited in the legend of Figure 1.

References

Decision letter

  1. Simon C Johnson
    Reviewing Editor; University of Washington, United States
  2. Matt Kaeberlein
    Senior Editor; University of Washington, United States
  3. Scott R Kennedy
    Reviewer; University of Washington, United States

In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.

Acceptance summary:

The manuscript details novel methodology for introducing functional mitochondria into mammalian cells, which work in both immortalized and primary cell lines. In addition, the authors provide data demonstrating a failure of mitochondrial transfer via simple co-incubation with mitochondria, an important observation. Overall, this is a promising new technique, and should be of high interest to researchers studying mitochondrial-nuclear crosstalk, mitonuclear compatibility, and related fields.

Decision letter after peer review:

Thank you for submitting your article "Stable transplantation of human mitochondrial DNA by high-throughput, pressurized mitochondrial delivery" for consideration by eLife. Your article has been reviewed by two peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by Matt Kaeberlein as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Scott R Kennedy (Reviewer #2).

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

We would like to draw your attention to changes in our revision policy that we have made in response to COVID-19 (https://elifesciences.org/articles/57162). Specifically, when editors judge that a submitted work as a whole belongs in eLife but that some conclusions require a modest amount of additional new data, as they do with your paper, we are asking that the manuscript be revised to either limit claims to those supported by data in hand, or to explicitly state that the relevant conclusions require additional supporting data.

Our expectation is that the authors will eventually carry out the additional experiments and report on how they affect the relevant conclusions either in a preprint on bioRxiv or medRxiv, or if appropriate, as a Research Advance in eLife, either of which would be linked to the original paper.

Summary:

Research into certain aspects of mitochondrial biology has been limited by a lack of robust methodology for introducing isolated mitochondria into recipient cells. To date, well-validated mitochondrial transfer methods have been highly specialized and extremely low throughput in nature. Sercel et al. describe a novel mitochondrial transfer technology that uses a plunger actuated mechanism to ballistically deliver isolated mitochondria into mtDNA-deficient recipient cells. They report evidence that transferred mitochondria are stably integrated into the recipient cell and compare this new approach to the previously described MitoCeption technology. The technique works in both immortalized and primary cell lines, with mitochondria persisting in numerous clones derived from individual cells. Furthermore, the authors provide data demonstrating a failure of mitochondrial transfer via simple co-incubation, an important observation.

Overall, this is an exciting new technique, and should be of high interest to researchers who study mitochondrial disease or the basic biology of mitochondria. However, there are a number of issues that should be addressed prior to publication.

Revisions for this paper:

1) Methodologic and technical details are insufficient in the current manuscript. We remind the authors that for this type of manuscript, "The article should fully describe the tool or resource so that prospective users have all the information needed to deploy it within their own work… methodological advances need to be comprehensibly described, along with details of the reagents and equipment, and their sources." Please provide detail, including improved diagrams and instructions, sufficient so that independent will be able to reproduce the approach. Where values may need to be empirically determined by other laboratories (such as how to determine the ideal voltage) details should be provided in how to approach the issue. In addition, authors need to note the approximate passage number/population doubling of the primary cells used and verify that they were similar between comparisons, as onset of senescence would undoubtedly have a dramatic impact of success in these assays. Figure legend and text also need more detail – for example, in Figure 4, it can be assumed that immune staining was used for TOM20 detection, and in section “ SIMR cells rescue ρ0 mitochondria network morphology and respiration” needs to state how mtDNA was imaged. Readers should not have to reference methods for these simple details.

2) Experiments showing stable incorporation of functional mitochondria were performed in uridine-free media to select for cells with mitochondria. Authors should demonstrate that mitochondria remain following passage in uridine-containing media. This data may in fact be present in the freeze-thaw culture experiments, but needs to be explicitly stated – it is not clear which media was used in those assays.

3) Additional information is needed regarding the mitochondrial isolation methodology, and data showing variability between mitochondrial preparations is necessary, rather than just technical replicates from the same isolation. It is impossible to know if the preparation used is an outlier in terms of performance, and it is important to know how reproducible the entire technique is. In regard to the technique, providing the kit used is simply not sufficient, particularly given that the brief description provided does not seem to match with the kit protocol available online. Further, one reviewer expressed concern that without these details we cannot be confident that contaminating donor cells weren't present in the isolated mitochondria, which may survive and outcompete mitochondrial deficient cells. It would also be useful to indicate, at least in the Discussion, what impact different methods of mitochondrial isolation are expected to have on success in these procedures.

4) The first data section, “ Mitochondrial delivery into transformed and primary cells”, should be re-worded. The data does not assess delivery INTO cells, as suggested by the prose; rather, the data presents co-association after 2 hours. The subsequent data demonstrates that actual delivery into cells does not correlate with the co-staining shown in this section, and the SEM data indicates that co-incubation simply results in an extracellular coating of mitochondria. The subsequent data clearly show a lack of actual transfer using co-incubation. I believe this is what the authors intended.

5) There are a few issues with Figure 3. In the titration experiments, the interpretation is not supported. The authors show that co-staining by flow cytometry does not mean mitochondria were taken into recipient cells, so in Figure 3G the right half of the panel is not useful and the 4-fold increase in efficiency is a misrepresentation. The data currently shown is “SIMR colonies per 104 cells that show co-staining with ds-RED”, not “recipient cells”. It is not clear that MitoPunch actually outperforms MitoCeption. The left half of the panel would suggest they perform similarly in this cell type. Truthfully, given the distinct properties of these two methods, it may not be reasonable to try to directly compare their relative efficacy in such a manner. It is sufficient to show they both work well and in a mitochondrial-mass dose-dependent manner, whereas co-incubation fails at all mitochondrial mass concentrations. Given the current state of the field, the new technique is still exciting, even if it has not been tested alongside MitoCeption in a way which allows direct head to head comparisons of efficacy. Future studies may delve deeper into the cell type, conditions, etc, where each technique best performs, but if the authors wish to claim here that MitoPunch is superior, additional data will be needed. It is assumed by the reviewer, but needs to be clarified/confirmed, that in the Figure 3G experiments the same cell number was used.

https://doi.org/10.7554/eLife.63102.sa1

Author response

Revisions for this paper:

1) Methodologic and technical details are insufficient in the current manuscript. We remind the authors that for this type of manuscript, "The article should fully describe the tool or resource so that prospective users have all the information needed to deploy it within their own work… methodological advances need to be comprehensibly described, along with details of the reagents and equipment, and their sources." Please provide detail, including improved diagrams and instructions, sufficient so that independent will be able to reproduce the approach. Where values may need to be empirically determined by other laboratories (such as how to determine the ideal voltage) details should be provided in how to approach the issue.

We included a new Figure 1—figure supplement 1 showing an annotated photograph of the MitoPunch device and also included new subsections in the Materials and methods detailing how to construct, tune, and operate the device as suggested.

In addition, authors need to note the approximate passage number/population doubling of the primary cells used and verify that they were similar between comparisons, as onset of senescence would undoubtedly have a dramatic impact of success in these assays.

We included these details in the revised text to note the approximate passage number of the BJ ρ0 cells used in the study, as suggested.

Figure legend and text also need more detail – for example, in Figure 4, it can be assumed that immune staining was used for TOM20 detection, and in section “ SIMR cells rescue ρ0 mitochondria network morphology and respiration” needs to state how mtDNA was imaged. Readers should not have to reference methods for these simple details.

We updated the figure legends and revised the Results text to include greater procedural details throughout the manuscript, including particular attention to our discussion of immunostaining, to clarify methodological details, as suggested.

2) Experiments showing stable incorporation of functional mitochondria were performed in uridine-free media to select for cells with mitochondria. Authors should demonstrate that mitochondria remain following passage in uridine-containing media. This data may in fact be present in the freeze-thaw culture experiments, but needs to be explicitly stated – it is not clear which media was used in those assays.

We indicated in the revised text that the SIMR clones were grown in uridine supplemented medium following their initial derivation as suggested.

3) Additional information is needed regarding the mitochondrial isolation methodology, and data showing variability between mitochondrial preparations is necessary, rather than just technical replicates from the same isolation. It is impossible to know if the preparation used is an outlier in terms of performance, and it is important to know how reproducible the entire technique is.

We performed additional experiments to demonstrate the reproducibility of the mitochondrial isolation method and the MitoPunch technique, as suggested. In short, we performed three triplicate MitoPunch experiments using three independent mitochondrial isolations and measured clone formation and mitochondrial mass from each preparation. We included this additional data as Figure 3—figure supplements 3 and 4 and also included relevant revised text in the Results section as suggested.

In regard to the technique, providing the kit used is simply not sufficient, particularly given that the brief description provided does not seem to match with the kit protocol available online. Further, one reviewer expressed concern that without these details we cannot be confident that contaminating donor cells weren't present in the isolated mitochondria, which may survive and outcompete mitochondrial deficient cells.

We provided additional details and clarification in the revised Materials and methods text regarding our mitochondrial isolation method, clearly describing the exact procedure used. We also performed an additional experiment represented in Figure 3—figure supplement 1 to demonstrate the lack of surviving donor cells in our mitochondrial preparations.

It would also be useful to indicate, at least in the Discussion, what impact different methods of mitochondrial isolation are expected to have on success in these procedures.

We developed the MitoPunch device and optimized operational procedures using mitochondria isolated by Dounce homogenization with standard mitochondrial isolation buffers (data not shown), and later adopted needle disruption and the Qiagen Qproteome kit to streamline the pipeline and improve reproducibility. We have not used other methods to isolate mitochondria for mitochondrial transfer, but we speculate that preparations generated in other ways would also yield SIMR clones. We included revised text in the Discussion section of the manuscript to address this point.

4) The first data section, “ Mitochondrial delivery into transformed and primary cells”, should be re-worded. The data does not assess delivery INTO cells, as suggested by the prose; rather, the data presents co-association after 2 hours. The subsequent data demonstrates that actual delivery into cells does not correlate with the co-staining shown in this section, and the SEM data indicates that co-incubation simply results in an extracellular coating of mitochondria. The subsequent data clearly show a lack of actual transfer using co-incubation. I believe this is what the authors intended.

We thank the reviewers for noting this discrepancy. We intended the prose to indicate the occurrence of mitochondrial association with recipient cells following delivery without an implication of the mechanism of co-localization. We revised the text to better represent the result and reflect our intended conclusion, as suggested.

5) There are a few issues with Figure 3. In the titration experiments, the interpretation is not supported. The authors show that co-staining by flow cytometry does not mean mitochondria were taken into recipient cells, so in Figure 3G the right half of the panel is not useful and the 4-fold increase in efficiency is a misrepresentation. The data currently shown is “SIMR colonies per 104 cells that show co-staining with ds-RED”, not “recipient cells”. It is not clear that MitoPunch actually outperforms MitoCeption. The left half of the panel would suggest they perform similarly in this cell type.

We agree with the reviewers that this result can lead to an unintended interpretation. We intended to show that the SIMR generation efficiency is greater for each cell that interacts with dsRed mitochondrial material by MitoPunch. To avoid confusion for readers, we removed this plot from the manuscript and revised the Results text.

Truthfully, given the distinct properties of these two methods, it may not be reasonable to try to directly compare their relative efficacy in such a manner. It is sufficient to show they both work well and in a mitochondrial-mass dose-dependent manner, whereas co-incubation fails at all mitochondrial mass concentrations. Given the current state of the field, the new technique is still exciting, even if it has not been tested alongside MitoCeption in a way which allows direct head to head comparisons of efficacy. Future studies may delve deeper into the cell type, conditions, etc, where each technique best performs, but if the authors wish to claim here that MitoPunch is superior, additional data will be needed. It is assumed by the reviewer, but needs to be clarified/confirmed, that in the Figure 3G experiments the same cell number was used.

Our intent was not to claim that MitoPunch is necessarily superior to MitoCeption, but to demonstrate that application of pressure during mitochondrial transfer enhances SIMR generation in different systems and also to highlight that the two methods have distinct use cases depending on the recipient cell type of interest. We included new text in the Discussion to clarify this point. We additionally clarified that the same number of cells were seeded for each replicate in Figure 3—figure supplement 5.

https://doi.org/10.7554/eLife.63102.sa2

Article and author information

Author details

  1. Alexander J Sercel

    Molecular Biology Interdepartmental Doctoral Program, University of California, Los Angeles, Los Angeles, United States
    Contribution
    Conceptualization, Data curation, Formal analysis, Funding acquisition, Validation, Investigation, Visualization, Methodology, Writing - original draft, Project administration
    Contributed equally with
    Alexander N Patananan
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-0749-2162

  2. Alexander N Patananan

    Department of Pathology and Laboratory Medicine, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, United States
    Contribution
    Conceptualization, Funding acquisition, Validation, Investigation, Methodology, Writing - review and editing
    Contributed equally with
    Alexander J Sercel
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-9458-9968

  3. Tianxing Man

    Department of Mechanical and Aerospace Engineering, University of California, Los Angeles, Los Angeles, United States
    Contribution
    Software, Formal analysis, Investigation, Methodology
    Competing interests
    No competing interests declared

  4. Ting-Hsiang Wu

    1. NanoCav, LLC, Culver City, United States
    2. NantBio, Inc, and ImmunityBio, Inc, Culver City, United States
    Contribution
    Software, Methodology
    Competing interests
    T.-H.W. was an employee of NanoCav, LLC, and is currently employed by NantBio, Inc and ImmunityBio, Inc.

  5. Amy K Yu

    Molecular Biology Interdepartmental Doctoral Program, University of California, Los Angeles, Los Angeles, United States
    Contribution
    Formal analysis, Investigation
    Competing interests
    No competing interests declared

  6. Garret W Guyot

    Department of Pathology and Laboratory Medicine, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, United States
    Contribution
    Investigation
    Competing interests
    No competing interests declared

  7. Shahrooz Rabizadeh

    1. NanoCav, LLC, Culver City, United States
    2. NantBio, Inc, and ImmunityBio, Inc, Culver City, United States
    3. NantOmics, LLC, Culver City, United States
    4. California NanoSystems Institute, University of California, Los Angeles, Los Angeles, United States
    Contribution
    Funding acquisition
    Competing interests
    S.R. is a board member of NanoCav, LLC, and employed by NantBio, Inc, ImmunityBio, Inc, and NantOmics, LLC.

  8. Kayvan R Niazi

    1. NanoCav, LLC, Culver City, United States
    2. NantBio, Inc, and ImmunityBio, Inc, Culver City, United States
    3. California NanoSystems Institute, University of California, Los Angeles, Los Angeles, United States
    4. Department of Bioengineering, University of California, Los Angeles, Los Angeles, United States
    Contribution
    Funding acquisition
    Competing interests
    K.R.N. is a board member of NanoCav, LLC, and employed by NantBio, Inc and ImmunityBio, Inc.

  9. Pei-Yu Chiou

    1. Department of Mechanical and Aerospace Engineering, University of California, Los Angeles, Los Angeles, United States
    2. California NanoSystems Institute, University of California, Los Angeles, Los Angeles, United States
    3. Department of Bioengineering, University of California, Los Angeles, Los Angeles, United States
    Contribution
    Software, Funding acquisition, Methodology
    Competing interests
    P.-Y.C. is a co-founder, board member, shareholder, and consultant for NanoCav, LLC, a private start-up company working on mitochondrial transfer techniques and applications.

  10. Michael A Teitell

    1. Molecular Biology Interdepartmental Doctoral Program, University of California, Los Angeles, Los Angeles, United States
    2. Department of Pathology and Laboratory Medicine, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, United States
    3. California NanoSystems Institute, University of California, Los Angeles, Los Angeles, United States
    4. Department of Bioengineering, University of California, Los Angeles, Los Angeles, United States
    5. Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research University of California, Los Angeles, Los Angeles, United States
    6. Department of Pediatrics, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, United States
    7. Jonsson Comprehensive Cancer Center, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, United States
    Contribution
    Conceptualization, Supervision, Funding acquisition, Methodology, Writing - review and editing
    For correspondence
    mteitell@mednet.ucla.edu
    Competing interests
    M.A.T. is a co-founder, board member, shareholder, and consultant for NanoCav, LLC, a private start-up company working on mitochondrial transfer techniques and applications.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-4495-8750

Funding

National Institutes of Health (T32CA009120)

  • Alexander J Sercel
  • Alexander N Patananan

National Institutes of Health (T32GM007185)

  • Alexander J Sercel

American Heart Association (18POST34080342)

  • Alexander N Patananan

National Institutes of Health (T32GM008042)

  • Amy K Yu

National Science Foundation (CBET 1404080)

  • Pei-Yu Chiou

National Institutes of Health (R01GM114188)

  • Pei-Yu Chiou
  • Michael A Teitell

Air Force Office of Scientific Research (FA9550-15-1-0406)

  • Pei-Yu Chiou
  • Michael A Teitell

National Institutes of Health (R01GM073981)

  • Michael A Teitell

National Institutes of Health (R01CA185189)

  • Michael A Teitell

National Institutes of Health (R21CA227480)

  • Michael A Teitell

National Institutes of Health (P30CA016042)

  • Michael A Teitell

CIRM (RT3-07678)

  • Michael A Teitell

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

We thank Rebeca Acin-Perez, Linsey Stiles, and Orian Shirihai of the UCLA Metabolomics Core for help with Seahorse XF Analyzer assays. We thank Zoran Galic, Alejandro Garcia, and Salem Haile of the UCLA Jonsson Comprehensive Cancer Center Flow Cytometry Core Laboratory and Felecia Codrea, Jessica Scholes, and Jeffrey Calimlim of the UCLA Broad Stem Cell Research Center Flow Cytometry Core for assistance with cellular analysis. We thank Laurent Bentolila and Matthew J Schibler of the UCLA Advanced Light Microscopy/Spectroscopy core for assistance with confocal microscopy. We also thank Emma Dawson, Lynnea Waters, Natasha Carlson, and Christopher Sercel for critical feedback and assistance with this manuscript.

Senior Editor

  1. Matt Kaeberlein, University of Washington, United States

Reviewing Editor

  1. Simon C Johnson, University of Washington, United States

Reviewer

  1. Scott R Kennedy, University of Washington, United States

Version history

  1. Received: September 15, 2020
  2. Accepted: January 12, 2021
  3. Accepted Manuscript published: January 13, 2021 (version 1)
  4. Version of Record published: February 5, 2021 (version 2)

Copyright

© 2021, Sercel et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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  1. Alexander J Sercel
  2. Alexander N Patananan
  3. Tianxing Man
  4. Ting-Hsiang Wu
  5. Amy K Yu
  6. Garret W Guyot
  7. Shahrooz Rabizadeh
  8. Kayvan R Niazi
  9. Pei-Yu Chiou
  10. Michael A Teitell
(2021)
Stable transplantation of human mitochondrial DNA by high-throughput, pressurized isolated mitochondrial delivery
eLife 10:e63102.
https://doi.org/10.7554/eLife.63102

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