Structural and Functional Diversity of Mitochondria Isolated from Different Cell Types

Abstract

Mitochondria are essential organelles responsible for energy production, autophagy, and apoptosis, and mitochondrial dysfunction has been implicated in various diseases affecting the heart, liver, and kidneys. Mitochondrial transplantation, wherein isolated mitochondria are administered into cells or tissues, has recently emerged as a promising therapeutic approach for restoring cellular functions by enhancing ATP generation and reducing oxidative stress. However, the characteristics and functional diversity of the mitochondria isolated from different cell types remain poorly understood. Here, we aimed to identify the optimal mitochondrial source for transplantation therapy by comparing mitochondria isolated from several mammalian cell types, including mesenchymal stromal, hepatic, muscular, and pluripotent stem cells. Mitochondria were isolated using a streptolysin O-based isolation method and characterized through particle size, zeta potential, protein content, and ATP content. The isolated mitochondria exhibited uniform morphology, negative surface charge, sufficient protein yield, and ATP content, indicating successful preparation of functionally competent organelles suitable for comparative analysis. The mitochondria derived from mesenchymal stromal cells exhibited the highest bioenergetic activity. Adding these mitochondria enhanced cellular proliferation, oxygen consumption, and resistance to oxidative stress in recipient cells. Collectively, these findings demonstrate that mitochondria isolated from autologous mesenchymal stromal cells possess superior bioenergetic properties, highlighting their potential as an optimal source for mitochondrial transplantation therapy and providing new insights into the design of mitochondria-based therapeutics.

INTRODUCTION

Mitochondria are dynamic organelles essential for maintaining cellular homeostasis.^1,2) In addition to producing ATP through oxidative phosphorylation, they regulate calcium signaling, lipid metabolism, redox balance, and apoptosis.³⁾ Their quantity and activity differ markedly among cell types, reflecting distinct metabolic demands.^4,5) Consequently, mitochondrial integrity is a key determinant of cellular function and cell survival. Mitochondrial dysfunction caused by genetic mutations, oxidative stress, or impaired quality control leads to decreased ATP generation and excessive reactive oxygen species (ROS) production, ultimately resulting in metabolic collapse and cell death.⁶⁾ These defects are implicated in diverse disorders, including cardiovascular, hepatic, and neurodegenerative diseases.⁷⁾ Moreover, age-related decline in mitochondrial turnover accelerates tissue degeneration and systemic metabolic imbalance.^8,9) Because of their central role in energy metabolism and cell fate regulation, mitochondria are promising therapeutic targets.

Recent advances have revealed that mitochondria are not static but exchangeable between cells, highlighting their potential as mediators of intercellular communication.^10–12) These insights have stimulated the development of innovative strategies that utilize mitochondria to restore or replace impaired organelles, thereby offering new directions for treating diseases associated with mitochondrial dysfunction.^6,13) Accordingly, three major approaches have been proposed to exploit the biological potential of mitochondria: (i) intercellular mitochondrial transfer: functional mitochondria are naturally exchanged between cells through tunneling nanotubes or gap junctions^14,15); (ii) vesicle-mediated mitochondrial delivery: mitochondria are encapsulated within extracellular vesicles or artificial lipid carriers^16,17); and (iii) mitochondrial transplantation: isolated mitochondria are directly introduced into recipient cells or tissues.^18–23) Each of these approaches harnesses the inherent bioenergetic and signaling functions of mitochondria to promote cellular recovery and tissue regeneration.⁶⁾ Among them, mitochondrial transplantation offers distinct advantages by enabling direct and controlled delivery of functional mitochondria.²⁴⁾ Preclinical studies have demonstrated that transplantation of isolated mitochondria restores ATP production, reduces oxidative stress, and improves tissue repair in models of ischemic injury,^18,19) liver damage,^20,21) and neurodegenerative disease.^22,23) By compensating for the dysfunction of damaged organelles, mitochondrial transplantation represents a conceptually unique and broadly applicable therapeutic platform for diseases associated with mitochondrial dysfunction.

Despite these promising findings, several challenges limit the clinical application of mitochondrial transplantation. A major issue is the heterogeneity of isolated mitochondria. Their morphology, membrane potential, and bioenergetic activity vary depending on the donor cell type, physiological conditions, and isolation method.^4,25,26) Recent reports have emphasized that the therapeutic efficacy of transplanted mitochondria strongly depends on their cellular origin and metabolic maturity.^19,27–29) Furthermore, large-scale production, preservation of stability, and immune compatibility remain unresolved issues, making it difficult to standardize mitochondrial preparations for therapeutic use.³⁰⁾ From a safety standpoint, the persistence of donor-derived mitochondrial DNA (mtDNA) in recipient cells raises concerns regarding potential genetic recombination and long-term cellular reprogramming.^31,32) Autologous transplantation using a patient’s own cells can mitigate these risks; however, an optimal cell source for obtaining functional mitochondria has not yet been established. Because mitochondria differ in their physicochemical and bioenergetic characteristics among cell types, a systematic comparison is essential to identify the origins that confer superior therapeutic potential.

In this study, we aimed to identify the optimal mitochondrial source for transplantation therapy by systematically characterizing mitochondria isolated from various mammalian cell types using a streptolysin O (SLO)-based isolation method.²⁶⁾ We evaluated the particle size, zeta potential, protein and ATP contents, and biological activities (including cellular uptake, proliferation, and cytoprotective effects) to provide a comprehensive foundation for optimizing mitochondrial transplantation strategies.

MATERIALS AND METHODS

Materials

SLO, Hoechst 33342, and hydrogen peroxide (H₂O₂) were obtained from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). Carboxyfluorescein succinimidyl ester (CFSE) was obtained from Cayman Chemical Co., Inc. (Ann Arbor, MI, U.S.A.). LysoTracker Red DND-99 was obtained from Thermo Fisher Scientific Inc. (Waltham, MA, U.S.A.). All other chemicals used were of the highest commercially available grade.

Cell Culture

The mouse mesenchymal stromal cell line C3H10T1/2 was obtained from Dr. Hiroki Kagawa (Kyoto Pharmaceutical University, Kyoto, Japan) and cultured in Dulbecco’s modified Eagle’s medium (DMEM; FUJIFILM Wako Pure Chemical Corporation) supplemented with 15% heat-inactivated fetal bovine serum (FBS; Thermo Fisher Scientific Inc.) and 1% penicillin–streptomycin (PS; FUJIFILM Wako Pure Chemical Corporation). The human mesenchymal stromal cell line UE7T-13 was obtained from the JCRB Cell Bank (Osaka, Japan) and cultured in low-glucose DMEM (Thermo Fisher Scientific Inc.) with 10% FBS and 1% PS. Human hepatocellular carcinoma HepG2 cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA, U.S.A.) and cultured in Eagle’s minimum essential medium (EMEM; FUJIFILM Wako Pure Chemical Corporation) supplemented with 10% FBS and 1% MEM non-essential amino acids (Nacalai Tesque, Inc., Kyoto, Japan). Mouse hepatocellular carcinoma Hepa1–6 cells were purchased from ATCC and cultured in DMEM supplemented with 10% FBS and 1% PS. The mouse myoblast cell line C2C12 was obtained from Professor Yoshikazu Higami (Tokyo University of Science, Tokyo, Japan) and cultured in DMEM supplemented with 10% FBS and 1% PS. The human-induced pluripotent stem cell (iPSC) line iPS72.3 was obtained from Professor James M. Wells (Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, U.S.A.) and cultured in mTeSR1 (StemCell Technologies Inc., Vancouver, BC, Canada). iPS72.3-derived mesenchymal progenitor cells (MPC) were differentiated from the iPS72.3 cells using the STEMdiff Mesenchymal Progenitor Kit (StemCell Technologies Inc.). All cells were maintained in a humidified incubator at 37°C with 5% CO₂.

Isolation of Mitochondria

Mitochondria were isolated from various cell types as previously described,²⁶⁾ with some modifications. Briefly, cells were cultured under standard conditions, detached with 2.5 g/L trypsin/1 mmol/L ethylenediaminetetraacetic acid solution (Nacalai Tesque, Inc.), and neutralized with complete media. After harvesting by centrifugation at 300 × g for 3 min, the cells were washed once with phosphate-buffered saline (PBS) and centrifuged again under the same conditions. The cell pellet was resuspended in HEPES-CH₃COOK buffer (115 mM CH₃COOK, 2.5 mM MgCl₂, 1 mM dithiothreitol, and 25 mM HEPES, pH 7.4), and SLO was added to a final concentration of 1 mg/L. The suspension was incubated on ice for 10 min to allow SLO binding to the plasma membrane, followed by centrifugation at 600 × g for 3 min at 4°C, and the resulting pellet was washed twice with Tris-sucrose buffer (250 mM sucrose, 0.5 mM ethylenediaminetetraacetic acid (EDTA), and 10 mM Tris–HCl, pH 7.4) to eliminate the residual SLO. To form SLO-induced small pores in the plasma membrane, the suspension was incubated at 37 °C for 10 min. To enlarge these pores and achieve selective disruption of the plasma membrane while minimizing mechanical damage to mitochondria, the suspension was gently pipetted 200 times per mL. The sample was centrifuged at 600 × g for 10 min at 4°C to remove unbroken cells, nuclei, and large cell debris. This centrifugation step was repeated until no visible pellet remained. The combined supernatants were further centrifuged at 7000 × g for 10 min at 4 °C to pellet the mitochondria. Pellets were resuspended in the appropriate buffer and proteins were quantified using the BCA assay (Nacalai Tesque, Inc.).

Western Blotting Analysis

Mitochondrial pellets isolated from 5 × 10⁶ cells of each human cell line were lysed in radioimmunoprecipitation assay buffer (Nacalai Tesque, Inc.) supplemented with Protease Inhibitor Cocktail Set III, EDTA-free, DMSO solution (×100; FUJIFILM Wako Pure Chemical Corporation) on ice. The lysates were centrifuged at 16000 × g for 15 min at 4°C, and the protein concentration in the supernatant was determined using a BCA protein assay kit. For immunoblotting, the entire lysate obtained from each mitochondrial pellet was mixed with 4× Laemmli sample buffer (250 mM Tris–HCl, 8% SDS, 40% glycerol, 0.04% bromophenol blue, pH 6.8), and 100 mM dithiothreitol was added immediately before use. The samples were boiled at 95°C for 3 min and loaded onto gels. Electrophoresis was performed using 12% polyacrylamide gels prepared with the TGX FastCast Acrylamide Kit (Bio-Rad Laboratories, Inc., Hercules, CA, U.S.A.) at 150 V. Proteins were transferred onto Immobilon-P polyvinylidene difluoride membranes (Merck Millipore, Burlington, MA, U.S.A.) at 100 V. The membranes were blocked with 5% bovine serum albumin in PBS containing 0.1% Tween-20 and incubated with a primary antibody against cytochrome c oxidase subunit IV (11242-1-AP; Proteintech Group, Inc., Rosemont, IL, U.S.A.) for 1 h at room temperature. After washing, the membranes were incubated with a horseradish peroxidase-conjugated anti-rabbit immunoglobulin G (IgG) secondary antibody (#7074; Cell Signaling Technology, Inc., Danvers, MA, U.S.A.) for 1 h at room temperature. Protein bands were visualized using Immobilon Western Chemiluminescent HRP Substrate (Merck Millipore) and detected using the iBright Imaging System (Thermo Fisher Scientific, Inc.). Band intensities were quantified using ImageJ (National Institutes of Health, Bethesda, MD, U.S.A.), and a regression curve was obtained between the total protein amount recovered from the same starting cell number and the corresponding cytochrome c oxidase (COX) band intensity.

Characterization of the Isolated Mitochondria

Mitochondrial ATP content was evaluated using an ATP assay kit (Dojindo Laboratories Inc., Kumamoto, Japan). Mitochondria isolated from 1 × 10⁷ cells were resuspended in 150 μL of PBS, and luminescence was measured using the GloMax Discover System (Promega Corp., Madison, WI, U.S.A.). For physicochemical measurements, mitochondria isolated from 1 × 10⁷ cells were resuspended in 1 mL of PBS. The cumulant diameter was measured by dynamic light scattering (DLS), and the zeta potential was determined by electrophoretic light scattering (ELS) using a particle analyzer (ELSZ-2000ZS; Otsuka Electronics Co., Ltd., Osaka, Japan).

Cell Proliferation Assay

C3H10T1/2 cells were seeded at a density of 4 × 10³ cells/well in 96-well plates and cultured overnight. Cell proliferation was evaluated in two experimental settings. For the donor comparison experiment, the medium was replaced with DMEM containing mitochondria isolated from C3H10T1/2, HepG2, C2C12, or iPS72.3 cells at a final protein concentration of 50 mg/L. For the dose–response experiment, the medium was replaced with DMEM containing mitochondria isolated from C3H10T1/2 cells at final protein concentrations of 0, 2, 10, or 50 mg/L. After 24 h, cell proliferation was assessed using Cell Counting Kit-8 (CCK-8; Dojindo Laboratories, Inc.), with background correction performed using wells containing mitochondria alone.

Flow Cytometry

C3H10T1/2 cells were seeded at a density of 8 × 10⁴ cells/well in a 48-well plate and incubated overnight. The medium was replaced with DMEM containing CFSE-labeled mitochondria (50 mg/L) isolated from C3H10T1/2, HepG2, C2C12, or iPS72.3 cells, and the cells were incubated for 24 h. After washing, the cells were treated with trypsin–EDTA to remove any surface-bound mitochondria and distinguish them from internalized mitochondria. The cells were fixed with 4% paraformaldehyde, and the percentage of CFSE-positive cells was measured using the BD FACS Lyric flow cytometer (Becton, Dickinson and Co., Franklin Lakes, NJ, U.S.A.) to evaluate mitochondrial uptake.

mtDNA Quantification by qPCR

UE7T-13 cells were seeded at 5 × 10⁴ cells/well in 12-well plates and, after overnight culture, mitochondria from C3H10T1/2 cells were added at 20 mg/L, and cells were collected at days 1, 2, 3, and 7 by trypsinization and washing with PBS. The cells were passaged every 3 d during the assay. Genomic DNA was extracted with the FastGene™ Genomic DNA Extraction Kit (NIPPON Genetics Co., Ltd., Tokyo, Japan). qPCR was performed on a CFX Connect Real-Time PCR System (Bio-Rad Laboratories, Hercules, CA, U.S.A.) using THUNDERBIRD™ Next SYBR^® qPCR Mix (Toyobo Co., Ltd., Osaka, Japan) under standard two-step cycling with melt-curve analysis. Primers targeted mouse Cox2 and human GAPDH with the following sequences: mouse Cox2 forward, 5′-AGACGAAATCAACAACCCCG-3′, reverse, 5′-GGCAGAACGACTCGGTTATCA-3′; human GAPDH forward, 5′-TTGGCTACAGCAACAGGGTG-3′, reverse, 5′-GGGGAGATTCAGTGTGGTGG-3′. Relative donor mtDNA abundance was calculated by the 2^–ΔΔCt method normalized to human GAPDH.

Confocal Microscopic Observation

C3H10T1/2 cells were seeded at a density of 2 × 10³ cells/well in 8-well chamber slides and cultured overnight. Mitochondria from C3H10T1/2 cells were fluorescently labeled with CFSE and added to recipient cells at a final mitochondrial concentration of 40 mg/L. At 1, 3, 6, and 12 h after addition, the cells were stained with LysoTracker Red DND-99 (for endogenous lysosomes) and Hoechst 33342 (for nuclei), and washed with Hanks balanced salt solution (HBSS). Live-cell images were acquired using a TCS SP8 confocal microscope (Leica Microsystems GmbH, Wetzlar, Germany) to assess the localization of exogenous mitochondria relative to lysosomes.

Oxygen Consumption Rate (OCR) Measurement

OCR of C3H10T1/2 cells was measured using a Seahorse XF96 Analyzer (Agilent Technologies, Inc., Santa Clara, CA, U.S.A.). The cells (3 × 10⁴ cells/well) were seeded in XF96 Cell Culture Microplates (Agilent Technologies, Inc.) and incubated overnight at 37°C in a humidified incubator with 5% CO₂. Isolated mitochondria were added to the culture medium at concentrations of 0, 10, and 50 mg/L and incubated for 24 h. On the day of the assay, the culture medium was replaced with Seahorse XF assay medium (supplemented with 10 mM glucose, 1 mM pyruvate, and 2 mM glutamine, pH 7.4) and the cells were incubated at 37°C in a non-CO₂ incubator for 30 min. OCR was measured via sequential injections of 0.5 μM oligomycin, 2 μM FCCP, and 0.5 μM rotenone/antimycin A. Basal respiration rate, maximal respiration rate, and ATP production were calculated using the Wave software (Agilent Technologies, Inc.).

Evaluation of Cytoprotective Effect

C3H10T1/2 cells were seeded at a density of 4 × 10³ cells/well in 96-well plates and cultured overnight. The culture medium was then replaced with fresh medium containing 700 μM H₂O₂, and the cells were incubated for 1 h. After exposure, the cells were washed once with PBS, and the medium was replaced with fresh medium containing isolated mitochondria from C3H10T1/2 cells at final mitochondrial concentrations of 0, 2, 10, or 50 mg/L. After 24 h, cell viability was quantified using the CCK-8 assay, with background correction performed using wells containing mitochondria alone. To corroborate the CCK-8 results, images were acquired at 24 h using a fluorescence microscope (BZ-X800; Keyence Corp., Osaka, Japan).

Statistical Analysis

Statistical analyses and graphing were conducted using the R software (version 4.5.2; R Foundation for Statistical Computing, Vienna, Austria). Dunnett’s test was used for multigroup comparisons against a single control. ANOVA and F-tests were not performed to avoid issues related to multiple comparisons. p Values are shown in the figures; p < 0.05 was considered statistically significant.

RESULTS

Physicochemical Characteristics of Mitochondria Isolated from Cells

To compare the physicochemical properties of mitochondria isolated from different cell types, we measured their particle sizes and surface charges using DLS and ELS. As shown in Fig. 1, the average hydrodynamic diameter of mitochondria isolated from various cells ranged from approximately 700 to 1000 nm (Fig. 1a). The zeta potential of all mitochondrial samples ranged from −20 to −15 mV (Fig. 1b), demonstrating that isolated mitochondria possessed comparable negative surface charges. These results suggest that mitochondria isolated using SLO exhibit physicochemical characteristics independent of the donor cell type.

Fig. 1. Physicochemical Characteristics of Mitochondria Isolated from Different Cell Types

Hydrodynamic diameter (a) and zeta potential (b) of mitochondria isolated using the SLO-based isolation method were measured using DLS and ELS, respectively. Data represent the mean ± standard deviation (S.D.) (n = 3).

Biological Characteristics of Mitochondria Isolated from Cells

To evaluate the biological characteristics of mitochondria isolated from different cell types, total mitochondrial protein and ATP content was quantified (Fig. 2). The amount of mitochondrial protein recovered per 10⁷ cells varied by cell type, with particularly low protein yield observed in mitochondria isolated from pluripotent stem cells (Fig. 2a). To support the use of pellet protein amount as a proxy for mitochondrial recovery, we performed immunoblotting of COXIV using mitochondrial pellets prepared from the same starting cell number and found an overall positive association between the recovered protein amount and the COXIV band intensity (Supplementary Fig. S1). Mitochondrial ATP content per 10⁷ donor cells also differed depending on the cellular origin (Fig. 2b). Mitochondria derived from mesenchymal stromal cells (C3H10T1/2 and UE7T-13) and mesenchymal progenitor cells (iPS-MPCs) exhibited higher ATP content than those from other cell types, indicating that the bioenergetic capacity of isolated mitochondria is cell type-dependent. When normalized to protein content, the ATP-to-protein ratio further emphasized this difference (Fig. 2c), and mitochondria from C3H10T1/2, UE7T-13, and iPS-MPC cells showed the highest ATP content. We next compared representative donor cell lines used in this study (C3H10T1/2, HepG2, C2C12, and iPS72.3) using a common recipient cell line. Flow cytometry analysis at 24 h showed that the majority of recipient cells were CFSE-positive after mitochondrial addition across all donor groups (Supplementary Fig. 2a). Under the same experimental conditions, mitochondria from C3H10T1/2 cells showed the largest increase in cell proliferation among the donor cell lines tested (Supplementary Fig. 2b). Together, these results indicate that mitochondrial recovery and ATP content vary among donor cell types and that functional outcomes after transplantation can differ depending on the donor origin.

Fig. 2. Protein Content and ATP Levels of Isolated Mitochondria

(a) Protein amount, (b) mitochondrial ATP level per 10⁷ donor cells, and (c) ATP-to-protein ratio were determined to evaluate the bioenergetic capacity of mitochondria isolated from different cell types. Data represent the mean ± S.D. (n = 3).

Persistence of Donor Mitochondrial DNA

Since mitochondria possess their own mtDNA, exogenously introduced mitochondria may persist within recipient cells for an extended period. To assess the persistence of donor-derived mitochondrial genes after uptake, mitochondria isolated from mouse C3H10T1/2 cells were added to human UE7T-13 cells, and the mouse Cox2 gene was quantified in the recipient cells. As shown in Fig. 3, the amount of donor-derived mtDNA gradually decreased over time but remained detectable for at least 7 d after mitochondrial addition. These findings suggest that donor mtDNA persists transiently in recipient cells after mitochondrial uptake. Considering the potential risk of genetic recombination or heteroplasmy caused by residual donor mtDNA, mitochondria from autologous sources are preferable for therapeutic applications. Based on these findings, we established an autologous model by using C3H10T1/2 cells as both the mitochondrial donor and recipient in subsequent studies to further evaluate mitochondrial function.

Fig. 3. Persistence of Donor-Derived Mitochondrial DNA in Recipient Cells

Mouse Cox2 mtDNA in human UE7T-13 cells after addition of mitochondria from C3H10T1/2 cells (20 mg/L) was quantified using qPCR at 1, 2, 3, and 7 d. Relative expression levels were normalized to human GAPDH. Data represent the mean ± S.D. (n = 3).

Uptake of Mitochondria

To visualize mitochondrial uptake, mitochondria isolated from C3H10T1/2 cells were fluorescently labeled with CFSE and added to the C3H10T1/2 recipient cells. Confocal microscopy revealed cytoplasmic CFSE fluorescence as early as 1 h after addition (Fig. 4), indicating cellular internalization of the added mitochondria. Endogenous lysosomes were labeled with LysoTracker Red, and a substantial fraction of the CFSE signal colocalized with LysoTracker Red-positive structures, suggesting involvement of the endolysosomal pathway in mitochondrial internalization. Notably, LysoTracker-negative CFSE puncta were also observed in the cytoplasm 12 h after addition, suggesting that some mitochondria-derived signals were released from the lysosomes and distributed within the cytoplasm.

Fig. 4. Intracellular Localization of Exogenous Mitochondria

C3H10T1/2 cells were incubated with CFSE-labeled mitochondria from C3H10T1/2 cells (40 mg/L) for 1, 3, 6, and 12 h. Cells were stained with LysoTracker Red and Hoechst 33342 for visualization. Confocal microscopy revealed time-dependent colocalization of CFSE and LysoTracker signals, indicating lysosomal processing of exogenous mitochondria. Scale bars = 20 μm.

Effects of Mitochondrial Addition on Cellular Function

The biological effects of mitochondrial addition were evaluated in C3H10T1/2 cells. As shown in Fig. 5, treatment with mitochondria isolated from C3H10T1/2 cells significantly enhanced cell proliferation in a concentration-dependent manner (Fig. 5a). Consistently, measurement of OCR revealed that mitochondrial addition increased basal and maximal respiration and ATP production in recipient cells in a mitochondrial dose-dependent manner (Fig. 5b), indicating that transferred mitochondria contribute to cellular metabolic activation. To further assess the cytoprotective potential of isolated mitochondria, cells were exposed to H₂O₂-induced oxidative stress. As shown in Fig. 6, H₂O₂ treatment markedly reduced cell viability, whereas addition of mitochondria attenuated this effect in a dose-dependent manner. Microscopic observations supported these findings, showing that mitochondria-treated cells maintained their cell density and morphological integrity compared with untreated controls. These results demonstrate that mitochondria isolated from mesenchymal stromal cells promote cell proliferation, enhance metabolic activity, and protect cells from oxidative stress, highlighting their potential utility in therapeutic mitochondrial transplantation.

Fig. 5. Effect of Mitochondrial Addition on Cell Proliferation and Metabolic Activity

(a) Proliferation of C3H10T1/2 cells treated with increasing concentrations of mitochondria from C3H10T1/2 cells (0, 2, 10, or 50 mg/L) for 24 h was measured using CCK-8. (b) OCR parameters, including basal respiration, maximal respiration, and ATP production, were determined using the Seahorse XF Analyzer after mitochondrial addition (0, 10, and 50 mg/L). Data represent the mean ± S.D. (n = 3–7). Statistical significance was evaluated using Dunnett’s test vs. 0 mg/L.

Fig. 6. Cytoprotective Effect of Mitochondria against Oxidative Stress

(a) Relative cell number of C3H10T1/2 cells after H₂O₂ exposure and mitochondria treatment. C3H10T1/2 cells were treated with H₂O₂ (700 μM) for 1 h, washed with PBS, and then cultured in medium containing mitochondria isolated from C3H10T1/2 cells (0, 2, 10, or 50 mg/L) for further 24 h. (b) Representative microscopic images of live cells after treatment of H₂O₂ and mitochondria. Scale bars = 200 μm. Data represent the mean ± S.D. (n = 4). Statistical significance was evaluated using Dunnett’s test vs. H₂O₂ (+).

DISCUSSION

Several mitochondria-based therapeutic strategies have been developed, including intercellular mitochondrial transfer, vesicle-mediated delivery, and transplantation of isolated mitochondria.^14–23) Although these approaches share the goal of restoring cellular bioenergetics and function, they differ markedly in controllability, reproducibility, and suitability for clinical translation.^30,33) Direct transplantation of isolated mitochondria offers distinct advantages over other strategies. Isolated mitochondria can be prepared in a defined and reproducible manner, and their purity, quantity, and activity can be standardized before administration.³⁴⁾ This enables accurate dosing and quality control, which are essential for pharmaceutical development. Moreover, isolated mitochondria can be stored and transported under cryopreserved conditions without requiring living donor cells,^35,36) thus simplifying logistics and manufacturing. In contrast, cell-mediated mitochondrial transfer or vesicle-based delivery depends heavily on biological variability and donor cell viability, limiting scalability and consistency.³⁰⁾ Therefore, among mitochondria-based therapeutic modalities, the use of isolated mitochondria represents the most practical and clinically applicable approach that provides a controllable and well-characterized platform for future therapeutic development.

Mitochondrial isolation critically affects the quality and functional integrity of recovered organelles. Conventional differential centrifugation or detergent-based approaches often yield mitochondria that are partially damaged or contaminated with other organelles, resulting in variability in bioenergetic measurements.^37,38) Meanwhile, the SLO-based isolation method enables gentle and reproducible release of intact mitochondria from various cell types by forming transient pores in the plasma membrane.²⁶⁾ This technique minimizes mechanical stress and maintains both outer membrane structure and membrane potential, ensuring high-quality preparations suitable for functional evaluation. In this study, an SLO-based isolation method was adopted to enable consistent isolation of mitochondria from diverse donor cells under standardized conditions. The obtained mitochondria exhibited comparable particle sizes and zeta potentials across all the cell types, indicating that the procedure efficiently recovered organelles with similar physical integrity (Fig. 1). Thus, the SLO-based method provides experimental consistency and exhibits high applicability for mitochondria-based therapeutic products.

Previous studies have reported that donor-derived mtDNA can persist in recipient cells for varying durations after transfer.^31,32,39,40) In this study, donor mtDNA remained detectable for at least 7 d following mitochondrial addition (Fig. 3), whereas other reports have described its persistence for over 30 d in mitochondria-deficient cells.³²⁾ Because prolonged retention of donor mtDNA may lead to heteroplasmy or genetic recombination, use of autologous mitochondria is preferable from a safety perspective. However, several studies have shown that exogenous mtDNA can disappear within a week,⁴⁰⁾ suggesting that persistence depends on the recipient cell type, culture conditions, and donor mitochondrial characteristics. From a practical perspective, large-scale preparations for therapeutic use may require allogeneic transplantation. Although mitochondria derived from non-autologous cells can retain their ATP-generating activity, their immunological and ethical implications must be carefully considered before clinical application.

In this study, we compared candidate sources of therapeutic mitochondria across several cell types: mesenchymal stromal cells, which are considered promising mitochondrial donors because of their high clinical feasibility^41,42); hepatic cancer cells and myogenic cells, for which preclinical reports of mitochondrial transplantation already exist^43,44); and iPSCs together with their mesenchymal progeny, in view of future autologous applications.⁴⁵⁾ Mitochondria isolated from iPS72.3 cells showed a notably lower yield than those from the other cell types (Fig. 2), indicating that undifferentiated iPSCs are difficult to use directly as a practical source for mitochondrial transplantation. As a supplemental validation for using pellet protein amount as a proxy for mitochondrial recovery, immunoblotting of COXIV in mitochondrial pellets prepared from the same starting cell number showed an overall positive association between recovered protein amount and COXIV band intensity (Supplementary Fig. S1). Furthermore, all cell types except C3H10T1/2 cells, UE7T-13 cells, and iPS-MPCs exhibited low mitochondrial ATP content (Fig. 2), which was consistent with previous reports that hepatic cancer cells,⁴⁶⁾ myoblasts,⁴⁷⁾ and iPSCs⁴⁸⁾ displayed a glycolysis-dominant metabolic phenotype with relatively low mitochondrial ATP-producing capacity. By contrast, mitochondria from mesenchymal-lineage cells (C3H10T1/2 cells, UE7T-13 cells, and iPS-MPCs) showed high recovery and ATP content (Fig. 2), suggesting that mesenchymal cells are more suitable as mitochondrial donors and that iPSCs should preferably be differentiated into mesenchymal progeny before mitochondrial collection. However, the maintenance of iPSCs and the establishment of iPS-MPCs require substantial time and cost, which limits their feasibility for broad clinical use.⁴⁹⁾ We therefore focused on mesenchymal stromal cells as a more clinically accessible donor population and selected C3H10T1/2 cells as a representative mesenchymal stromal cell source for subsequent experiments.

We also compared representative donor cell lines (C3H10T1/2 cells, HepG2 cells, C2C12 cells, and iPS72.3 cells) used in this study for their ability to promote proliferation in a common recipient cell line. Although all transplanted mitochondria were efficiently internalized, the pro-proliferative effect was greatest for C3H10T1/2-derived mitochondria (Supplementary Fig. 2), supporting the notion that C3H10T1/2 cells provided the most suitable mitochondrial donors among the cell lines tested.

The intracellular fate of transplanted mitochondria appears to be context-dependent. Prior work has shown that mitochondria added to recipient cells colocalize with lysosomes as early as within 0.5 h and subsequently exit the endolysosomal compartments to fuse with the endogenous mitochondrial network.⁵⁰⁾ By contrast, another study reported that grafted mitochondria are predominantly processed by mitophagy without detectable fusion, with mitophagy acting as a trigger for mitochondrial biogenesis and therapeutic benefits, deviating from the classical “engraft-and-fusion” paradigm.⁴⁰⁾ Determinants likely arise on both sides, the donor and the recipient. On the donor side, mitochondria from certain cell sources express factors that attenuate recipient mitophagy, thereby favoring persistence and heteroplasmy.⁵¹⁾ On the recipient side, mtDNA-deficient cells retain donor-derived mtDNA longer than their mtDNA-replete counterparts, indicating that the recipient’s mitochondrial genotype and physiological state also influence persistence.³²⁾ In our system, exogenous mitochondria were consistently trafficked into lysosomes from the earliest time points for 12 h. Nevertheless, LysoTracker-negative mitochondria-derived fluorescence was still observed in the cytoplasm at 12 h, suggesting that a fraction of the transferred material may have evaded lysosomal degradation and potentially contributed to the observed functional effects.

The functional impact of mitochondria on recipient cells has been widely reported, and several studies have shown that these effects vary depending on the donor and recipient cell types and cellular state.^29,32,52,53) Nevertheless, most mitochondria share fundamental biological properties that contribute to enhanced cell proliferation and antioxidant defense, regardless of their origin.²⁹⁾ Consistent with these reports, our data confirmed that mitochondria isolated from C3H10T1/2 cells exerted similar proliferative and cytoprotective effects (Figs. 5, 6). These findings suggest that although mitochondrial bioenergetic efficiency may differ among cell types, their core functions supporting cell survival and redox balance are largely conserved, providing a common therapeutic potential across different mitochondrial sources.

In conclusion, as a representative mesenchymal stromal cell line selected in this study, C3H10T1/2-derived mitochondria showed relatively high recovery, ATP content, and pro-proliferative activity, and dose-dependently enhanced the bioenergetic status of recipient cells while protecting them against oxidative stress. These findings provide a practical basis for selecting and preparing functional mitochondria for transplantation; however, establishing whether similar benefits extend to other mesenchymal stromal cell sources will require additional studies to confirm their utility and to support broader generalization, particularly under different experimental conditions.

Acknowledgments

This work was supported by a Grant-in-Aid for Scientific Research (B) (Grant Numbers: 23H03749 and 23K28437) and a Grant-in-Aid for Challenging Research (Exploratory) (Grant Number: 23K18595) from the Japan Society for the Promotion of Science (JSPS), the Canon Foundation, the Leading Pioneers Science Foundation, the Greater Tokyo Innovation Ecosystem (GTIE) program supported by the Ministry of Education, Culture, Sports, Science and Technology (MEXT) and the Japan Science and Technology Agency (JST), and the Top Researcher Development Support Program of Tokyo University of Science. We thank Professor James M. Wells (Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, U.S.A.) for providing iPS72.3 cells.

DECLARATIONS

Conflict of Interest

The authors declare no conflict of interest.

Use of Generative Artificial Intelligence (AI)

We used ChatGPT (OpenAI; accessed October–November 2025) to assist with language editing and manuscript refinement; the tool was used solely to improve grammar and readability and to draft figure legends, and was not used to generate scientific content or analyze data.

Supplementary Materials

This article contains supplementary materials.

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