All protocols use sterile techniques in a Class II, Type A2 laminar flow hood. All culture plastic materials and surgical scalpel are sterile and disposal. Human skin tissue was donated by a young healthy man who signed informed consent. Skin tissue was surgically collected in outpatient clinical center and dropped to a 50 ml disposal sterile centrifuge tube containing 15 ml of sterile 0.9% sodium chloride injection. Skin (0.5 cm × 2 cm) was washed with cold sterile saline. Fat tissue was trimmed. The skin was cut to small pieces using a sterile disposal scalpel in a sterile disposal 100 mm culture dish. All tissue pieces and saline were transferred into a sterile glass chamber with cap and stir bar. 4.5 ml of 3% collagenase type 3 (Worthington Biochemical Corporation, Lakewood, NJ, USA) was added to the chamber. The final volume was 20 ml. The chamber was placed on a magnetic stirrer at speed 150 rpm in 37˚C oven for digestion. The tissue was digested for 5 hours with gentle stirring till tissue piece disappear. The liquid was transferred into a sterile 50 ml centrifuge tube and centrifuged for 5 minutes at 400 g. Aspirate the supernatant. Add 20 ml pre-warmed (37˚C) complete alpha minimum essential media (alpha MEM) (GIBCO, Carlsbad, CA, USA) containing 5% human platelet lysate (HPL) (Biological Industries, Cromwell, CT, USA) and 0.05 mg/ml gentamicin (GIBCO, Carlsbad, CA, USA). Re-suspend cell pellet and transfer cell suspension to a 182 cm²-flask. The flask was cultured at 37˚C with 5% CO₂ incubator overnight. The medium and floating cells was aspirated next day. 20 ml fresh medium was added to the flask. The flask was returned to the incubator for culture with medium refresh every 3 - 4 days. When cells growth was 80% confluent, cells were sub-cultured to new flasks by 1:10 dilution. The primary fibroblasts of second and third passages were collected, re-suspended in Nutrifreez D10 cryopreservation medium (Biological Industries, Cromwell, CT, USA), aliquoted to 1ml containing 1 × 10⁶ cells in cryovials, frozen and stored in liquid nitrogen.

Human mammary adenocarcinoma cell line MCF-7, mouse mammary adenocarcinoma cell line 4T1 and mouse mammary epithelium cell line EpH4-Ev were purchased from American Type Culture Collection (ATCC) (Manassas, VA, USA). MCF-7, 4T1 and human primary fibroblasts were recovered from liquid nitrogen and cultured in alpha MEM containing 10% fetal bovine serum (FBS) (GIBCO, Carlsbad, CA, USA). EpH4-Ev cells were culture in alpha MEM containing 10% FBS and 1.2 µg/mL puromycin (Sigma Aldrich, St. Louis, MO, USA). NSC-34 is a hybrid cell line, produced by fusion of motor neuron enriched, embryonic mouse spinal cord cells with mouse neuroblastoma. NSC-34 was purchased from Cedarlane corporation (Ontario, Canada) and cultured in Dulbecco’s modified eagle medium (DMEM) (GIBCO, Carlsbad, CA, USA) containing 10% FBS. When cells grew to 80% full in flask, they were digested with TrypLE expression solution (GIBCO, Carlsbad, CA, USA) and sub-cultured at 37˚C and 5% CO₂.

Mitochondrial were isolated by differential centrifugation following a previously described protocol [30 - 32]. All reagents were sterile. Cells were harvested with TrypLe expression solution and pelleted by centrifugation for 5 minutes at 400 ×g and at 4˚C. After aspiration of the solution and media, cell pellet was re-suspended in ice-cold 300 mM sucrose mitochondrial isolation buffer (MIB) (Sigma Aldrich, St. Louis, MO, USA) and homogenized by bead beating (Bead Ruptor 12, Omni International homogenizer company (Kennesaw, GA, USA). The cell lysate was centrifuged for 10 minutes at 700 ×g and at 4˚C. Then the supernatant was transferred to new centrifugation tubes and centrifuged for 10 minutes at 9000 ×g and at 4˚C. The supernatant was removed. The mitochondrial pellet was re-suspended with 240 mM sucrose mitochondrial respiration buffer (MRB) (Sigma Aldrich, St. Louis, MO, USA). In order to determine the number of isolated mitochondria, 10 µl of JC-1-stained mitochondria was placed on glass slide and covered with 22 mm × 22 mm coverslip (area 4.84 × 10⁸ µm²). The mitochondria were observed through 20x object lens, taken photo pictures in five fields and counted mitochondrial number each picture by Cellsense software of Olympus IX83 fluorescent microscope (Olympus, Tokyo, Japan). Object area each picture was calculated by using the scale bar (µm) in the pictures. Number of mitochondria in 10 µl was calculated as the following: (coverslip area 4.84 × 10⁸ µm² ÷ object area in µm² per picture) × the average of mitochondrial number per picture. Then total isolated mitochondria number was calculated [30 , 33].

Mitochondrial protein content can be used to estimate mitochondrial number [33]. The mitochondrial pellets were lysed by RIPA buffer (Thermofisher Scientific, Waltham, MA, USA). The mitochondria were shaken gently for 30 minutes on ice. Then the mitochondrial lysate was centrifuged at 14,000 ×g for 10 minutes to pellet debris. The supernatant was transferred to 1.5 ml vial for protein content measurement. The protein was quantitated by Pierce BCA protein assay kit (Thermo Scientific, Rockford, IL, USA). The procedure is seen in the protocol of the assay kit. In brief, pipette 25 µl of standard or sample to the replicate wells, add 200 µl of BCR working reagent to each well and mix plate thoroughly for 30 seconds, incubate the plate at 37˚C for 30 minutes, cool down the plate to room temperature, and measure the absorbance at 562 nm on a plate reader. The protein concentrations of samples were calculated by four-parameter logistic curve.

20,000 primary fibroblasts were cultured at glass bottom cell culture dishes overnight. Mitochondria were isolated from 5 × 10⁶ fibroblasts. fibroblasts in dishes and the mitochondria in 1.5 ml Eppendorf centrifugation tubes were fixed with 4% paraformaldehyde solution for 15 minutes at room temperature. The rabbit anti-human HLA-ABC polyclonal antibody (Thermofisher Scientific, Waltham, MA, USA) reacts to all types (HLA-A, -B and -C) of HLA I antigen. The brief procedure of immunofluorescent staining is as following: wash twice with 1x phosphate buffered saline (PBS), block with 5% goat serum in 1 × PBS, wash once, incubate with 200 µl 1:400 dilution of rabbit anti-human HLA-ABC polyclonal antibody overnight at 4˚C, wash 3 times with 1 × PBS, incubate with goat anti-rabbit IgG-Alexa 488 for 1 hour at room temperature, wash for 3 times with 1 × PBS, add 300 µl of 1 × PBS to the dishes and tubes, re-suspend the mitochondria in 1.5 ml Eppendorf tubes and add 100 µl of the mitochondria to the glass area of new glass bottom dishes, observe fluorescence under Olympus IX83 fluorescent microscope. All pictures were taken with 1 second exposure.

Mitochondrial membrane potential generated by proton pumps is an essential component in the process of energy storage during OXPHOS. Membrane potential dependent dyes such as JC-1(5,5’,6,6’-tetrachloro-1,1’,3,3’-tetraethylbenzimidazolocarbocyanine iodide) and MitoTracker dyes (rosamine- or cyCarbocyanine-based probes) have been used to stain mitochondria and monitor mitochondrial potential. For JC-1 staining, mitochondria were stained with mitochondria staining kit (Sigma CS0390, St. Louis, MO, USA). The brief procedure is as follows: Mix 25 µl of 200 × JC-1 Stock Solution in 4 mL of ultrapure water in a test tube. Close the test tube and mix solution by inversion or vortex test tube briefly. Incubate test tube for 2 min at room temperature to completely dissolve JC-1. Open the test tube and add 1 ml of JC-1 Staining Buffer 5×. Mix by inversion. Then, mix the staining mixture with an equal volume of complete medium for cell growth. Aspirate growth medium from flask and overlay cells with the above mixture. Add 0.2 - 0.4 ml of the mixture per cm² of growth surface. Incubate cells for 20 minutes at 37˚C in humidified atmosphere containing 5% CO₂. Aspirate the mixture. Wash the cells twice with cold growth medium. Fluorescence is observed by Olympus IX83 fluorescent microscope. In cells which maintain electrochemical potential gradient, the dye concentrates in mitochondria, where it forms bright red fluorescent aggregates (J-aggregates). If cells failed to maintain membrane potential, the JC-1 is dispersed through the entire cells resulting in a shift from red to green fluorescence (JC-1 monomers).

For MitoTracker dyes, culture media were aspirated. Cells were covered with fresh media containing 150 nM of MitoTracker Orange or Red dyes (Thermofisher Scientific, Waltham, MA, USA).and cultured for 30 minutes at 37˚C and 5% CO₂. Then, staining solution was removed. Cells were washed once, covered with fresh media, and observed under fluorescent microscope.

CellLight Mitochondria-RFP or -GFP, BacMam 2.0 were purchased from Invitrogen (Carlsbad, CA, USA). They are fluorescent and the leader sequence of E1 alpha pyruvate dehydrogenase (in mitochondrial matrix) fusions that provide accurate and specific targeting to mitochondria in live-cell mitochondrial imaging. Cells grew on culture dishes to 70% confluence. The appropriate volume of CellLight reagent for the number of cells were calculated as the protocol of the products. The volume of CellLight mitochondria-RFP or -GFP were added to cells. The cells were incubated at 37˚C at least 16 hours and observed by Olympus IX83 fluorescent microscope. Averaged number of mitochondria per fibroblast was determined by Cellsense software. In order to test mitochondrial transfer between adenocarcinoma cell MCF-7 and fibroblasts, mitochondria of MCF-7 and fibroblasts were labelled with CellLight Mitochondria-RFP or -GFP, respectively, for at least 16 hours. The media were removed. Cells were washed twice with prewarmed fresh media and dis-attached with TrypLE express reagent. Same number of MCF-7 and fibroblasts were mixed and co-cultured at 37˚C and 5% CO₂ for 24 hours. The fluorescence in cells was observed by Olympus IX83 fluorescent microscope.

It has been reported that long-term treatment of cells with low doses (0.1 - 2 µg/ml) of ethidium bromide (EtBr), an inhibitor of DNA/RNA synthesis, specifically suppresses the replication and transcription of extrachromosomal genetic components such as mtDNA without affecting nuclear DNA replication and transcription [34]. NSC-34 cells were maintained in the complete DMEM medium containing 2 µg/ml EtBr and sub-cultured every week with medium refresh every 3 - 4 days. After 3 months, total DNA of NSC-34 was isolated with QIAamp DNA mini kit (Qiagen, Hilden, Germany). MT-CO1 and MT-ND1 were tested by PCR. PCR was performed in a total 25 µl volume including 2 µl of PCR Master mix (Thermofisher Scientific, Waltham, MA, USA), 0.5 µl of 100 µM forward and reverse primers and 200 ng DNA template. The primers used for amplification of MT-CO1 were 5’-CTAGCCGCAGGCATTACTATAC-3’ and 5’-TGTCAAGGGATGAGTTGGATAAA-3’. The primers for MT-ND1 were 5’-GCCGTAGCCCAAACAATTTC-3’ and 5’-CGTAACGGAAGCGTGGATAA-3’. Mouse β-actin (ACTB) was used as endogenous gene control. The primers for ACTB amplification were 5’-CTGAGTCTCCCTTGGATCTTTG-3’ and 5’-AGGGCAGGTGAAACTGTATG-3’. The amplification procedure included initial DNA denaturing at 95˚C for 3 minutes, then 35 cycles of denaturing 30 seconds at 95˚C, primer annealing 30 seconds at 50˚C and 60 seconds of extension at 72˚C, and final extension of 10 minutes at 72˚C in a T100 Thermal Cycle (Bio-Rad, Hercules, CA, USA). PCR products were run on a 2% agarose gel and imaged by ethidium bromide fluorescence.

Mitochondria were isolated from mouse NSC-34 cells. The mitochondria (from 1 × 10⁶ cells) were co-cultured with 1 × 10⁵ NSC-34 ρ˚ cells (Mitochondria from 10 NSC-34 cells + 1 NSC-34 ρ˚ cell). Normal NSC-34 was as control. After 24 hours of co-culture, the media containing the mitochondria were removed. Cells were washed twice, refilled with fresh complete DMEM, and continuously cultured. DNA and RNA of cells were isolated at 1, 3 and 6 days, respectively, after transplantation for PCR and real time PCR examination. For imaging of mitochondrial transplantation, mitochondria of NSC-34 cells and primary fibroblasts were labelled with MitoTracker Red, isolated and co-cultured with NSC-34 ρ˚ cells for 24 hours (Mitochondria from 10 NSC-34 cells or fibroblasts + 1 NSC-34 ρ˚ cell). After removal of the media containing the labelled mitochondria, cells were observed under fluorescent microscope.

We used qPCR to measure mRNA levels of mitochondrial and glycolysis-associated genes. The detailed protocol was published previously [28]. We examined mitochondrial OXPHOS genes, MT-CO1 and MT-ND1, and glycolysis-associated genes HK2, SLC2A1and LDHA which catalyzed the reduction of pyruvate by NADH to form lactate. All gene expression quantification was performed with TaqMan Gene Expression Assay, a proven 5’ nuclease-based real-time PCR chemistry. Primers and probes (PrimeTime Mini qPCR assay) were synthesized by Integrated DNA Technologies (IDT, Coralville, Iowa) (Table 1). β-actin (ACTB) was used as endogenous gene control to normalized PCRs for the amount of RNA added to the reverse transcription reactions. Probes contain at the 5’ end the FAM (6-carboxy fluorescein) as a fluorescent reporter dye, and internal and at 3’ end the ZEN™/Iowa Black FQ as fluorescent double quenchers. The qPCR reaction was performed with 7900 HT real time PCR system (Applied Biosystems, Grand Island, NY). Standard mode ran as 2 minutes at 50˚C and 10 minutes at 95˚C, and 40 cycles [15 seconds at 95˚C (denaturing) and 1 minute at 60˚C (annealing/extending)]. Target gene expression was determined by relative quantification which related signal of the target transcript in an experimented group to that of a control. We analyzed relative quantification with the RQ Manager 1.2.1 software (Applied Biosystems, Grand Island, NY, USA).

The Institutional Animal Care and Use Committee (IACUC) protocol was submitted and approved by Louisiana State University School of Veterinary Medicine. Female mice were 16 - 18 grams (average 17.3 g) and 8 weeks old. 1 × 10⁵ mouse mammary adenocarcinoma 4T1 cells in 0.2 ml 0.9% NaCl was injected subcutaneously into each mouse. When tumor grew to 1cm diameter, 0.2 ml of isolated mitochondria (5 × 10⁷ mitochondria) from 1.0 × 10⁶ EpH4-Ev cells were directly injected into tumor. The mitochondria of normal epithelial EpH4-Ev were pre-labelled with MitoTracker orange dye, isolated and stored in MRB on ice. After 24 hours, tumor was biopsied and smeared on glass slides. Tumor smearing slides were observed under fluorescent microscope to check whether the labeled mitochondria entered into 4T1 tumor cells. To examine inhibition of normal mitochondria to tumor cells, mixture of 1 × 10⁵ 4T1 and mitochondria (5 × 10⁷ mitochondria) cells in 0.2 ml of MRB was injected subcutaneously into the dorsal cervical area of mouse (1 tumor 4T1 cell + mitochondria from 10 EpH4-Ev cells). Control group mice were only injected with 1 × 10⁵ 4T1 cells in 0.2 ml of MRB per mouse. Control group and mitochondria-treated group have10 and 11 mice, respectively. At 18 days post-injection, mice were sacrificed and necropsied. Tumor mass and organs including lungs, liver, heart, spleen, and kidneys were removed and weighed. All tissues were fixed in 4% formalin solution for histological study.

Student’s t-test was used to test statistical significance. p-value less than 0.05 was judged to be of statistical significance.

The life span of the human primary fibroblasts is approximate 80 days. The double time of growth is 40 ± 6 hours. We labelled mitochondria of fibroblasts with CellLight Mitochondria-GFP and counted number of mitochondria per fibroblast in 30 different fields by Cellsense software of Olympus IX83 fluorescent microscope. There are mitochondria of 337 ± 80 (mean ± standard deviation) per human fibroblast (Figure 1). Mitochondrial viability is rapidly and easily assessed using mitochondrial fluorescent probes [24 , 25 , 33]. We find that the isolated mitochondria actively concentrate JC-1 and form bright red fluorescent aggregates (J-aggregates) (Figure 2). This finding suggests that the isolated mitochondria are viable and maintain electrochemical potential gradient. Total (1.43 ± 0.33) × 10⁹ of mitochondria could be isolated from 1 × 10⁷ fibroblasts which totally have (3.37 ± 0.8) × 10⁹ mitochondria in cells. The mitochondrial isolation protocol can obtain 42.4% [(1.41 × 10⁹) ÷ (3.37 × 10⁹) × 100%] of fibroblast mitochondria. Mitochondrial protein content is an alternative method to estimate mitochondrial number [33]. The (1.43 ± 0.33) × 10⁹ of mitochondria that isolated from 1 × 10⁷ fibroblasts have 190 ± 45 µg protein content. Both the fibroblast number and mitochondrial protein content can estimate number of isolated mitochondria.

Green fluorescence is found on cell surface of human fibroblasts. The distribution of green fluorescence is uneven (Figure 3(b)). The finding suggests HLA class I antigens mainly express on cell surface of human fibroblasts. The isolated mitochondria have no green fluorescent staining (Figure 3(d)). These results suggest that mitochondria of human fibroblasts don’t express HLA antigens and may be used for allogeneic mitochondrial transplantation without HLA antigen match.

CellLight Mitochondria-GFP-labelled fibroblasts were co-cultured with CellLight Mitochondria-RFP-labelled MCF-7 for 24 hours. Mitochondria moving between fibroblasts (green) and MCF-7 cells (red) result in orange fluorescence in some cells (arrow in Figure 4).

After treatment of NSC-34 with 2 µg/ml EtBr for 3 months, PCR shows there is no amplification of mitochondrial genes MT-CO1(490 bp) and MT-ND1(445 bp). The treatment has no effect on genomic gene ACTB amplification (298 bp) (Figure 5). The results suggest long term of EtBr-treatment depleted mtDNA of NSC-34 cells. NSC-34 ρ˚ cells grow much slower than parent NSC-34. The growth of NSC-34 ρ˚ cells is approximate 10% of parent NSC-34. Isolated MitoTracker red-labelled mitochondria from NSC-34 cells or primary fibroblasts were co-cultured with NSC-34 ρ˚ cells. At 24 hours post-culture, mitochondria with red fluorescence are observed in NSC-34 ρ˚ cells (Figure 6(a), Figure 6(b)). These results suggest that isolated autologous and xenogeneic mitochondria transplant to the defective NSC-34 ρ˚ cells.

After co-culturing of NSC-34 ρ˚ cells with the isolated NSC-34 mitochondria for 24 hours, PCR shows that MT-CO1 DNA genes are amplified in NSC-34 ρ˚ cells. Moreover, MT-CO1 remains amplification at 3 and 6 days post-mitochondrial transplantation (Figure 7). Amplification of genomic gene ACTB is not changed by mitochondrial transplantation (Figure 7). Levels of gene expression are calculated as the formula: (gene expression of NSC-34 ρ˚ cells ÷ gene expression of NSC-34 cells) × 100%. qPCR shows that NSC34 ρ˚ cells have very low levels of mRNA expression of mitochondrial genes MT-CO1 and MT-ND1 (approximately 10% of parent NSC-34 cells), but regain mRNA expression to the levels of normal NSC-34 at 3 days post-mitochondrial transplantation (Figure 8). The expression of glycolytic genes, HK2, LDHA and SLC2A1, is upregulated in NSC34 ρ˚ cells. These suggest NSC-34 ρ˚ cells with defective mitochondria increase aerobic glycolysis to produce cell energy ATP. Mitochondrial transplantation downregulates expression of the glycolysis-related genes. The levels of HK2, LDHA and SLC2A1 gene expression return to normal at 3 days post-transplantation of NSC 34 mitochondria (Figure 9). These results suggest that the isolated NSC-34 mitochondria can effectively replenish mitochondrial DNA and rescue aerobic respiration of NSC 34 ρ˚ cells. In this study, 10 NSC-34 cells’ mitochondria can rescue 1 NSC 34 ρ˚ cells with defective mitochondria.

We find that xenogeneic mitochondria from human fibroblasts successfully enter to NSC 34 ρ˚ cells (Figure 6(b)). However, we haven’t examined the expression of human mitochondrial genes and glycolysis-associated genes in mouse NSC 34 ρ˚ cells. More studies remain to be done.

At 24 hours post-injection of MitoTracker orange-labelled mitochondria of normal EpH4-Ev cells into tumor mass, orange fluorescence was observed in 4T1 cells (Figure 10). This result suggests that the mitochondria of EpH4-Ev cells successfully transplant into tumor 4T1 cells. At 18 days of experiment, mice were necropsied. Tumor weight in the group that received normal EpH4-Ev mitochondria is not different from tumor weight of the control group (p > 0.05). Gross examine shows all mice have lung metastases in both groups. However, lung weight of the group that received mitochondria is less than that of the control group even though the difference is not quite significant (p = 0.06) (Table 2). These results suggest that exogeneous normal mitochondria might decrease metastatic burden in lungs.

Subcutaneous injection of mitochondria [5 × 10⁷ mitochondria (6.6 µg protein) per mouse] and MRB (0.2 ml/mouse) didn’t cause any side reaction in mice. The doses in mice can be converted to doses of human based on body surface area as the formula: dose of human per kilogram (kg) body weight = mouse dose per kg × 0.081 [35 , 36]. In this study, mouse averaged weight is 17.3 g. The doses of mice are 0.38 mg/kg [0.0066 mg (mitochondrial protein) ÷ 0.0173 kg mouse weight], or 2.89 × 10⁹ mitochondria/kg (5 × 10⁷ mitochondria ÷ 0.0173 kg mouse weight). The equivalent of human doses will be: 0.031 mg/kg (0.38 mg/kg in mice × 0.081) in protein or 2.34 × 10⁸ mitochondria/kg (2.89 × 10⁹ in mice × 0.081). These doses can be used as starting dose for clinical trial. Subcutaneous injection of 0.2 ml of MRB is safe for mice. Conversion of the MRB volume to human will be 2 - 5 ml intramuscular injection per site or <250 ml intravenous injection [35].

*mean ± standard deviation (number of mice); &Student’s t-test, less than 0.05 was judged to be of statistical significance.