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Developmental Biology

Mitochondrial Transfer from Mouse Adipose-Derived Mesenchymal Stem Cells into Aged Mouse Oocytes

Published: January 6, 2023 doi: 10.3791/64217


Due to the decline in the quantity and quality of oocytes related to age, the fertility of women over 35 years of age has declined sharply. The molecular mechanisms that maintain oocyte quality remain unclear, thus it is difficult to increase the birth rate of women over 35 years old at present. Oocytes contain more mitochondria than any type of cell in the body, and any mitochondrial dysfunction can lead to reduced oocyte quality. In the 1990s, oocyte cytoplasmic transfer resulted in great success in human reproduction but was accompanied by ethical controversies. Autologous mitochondrial transplantation is expected to be a useful technique to increase the quality of oocytes that have decreased due to age. In the present study, we used adipose-derived stem cells from aged mice as a mitochondria donor to increase the quality of oocytes of aged mice. Further development of autologous mitochondrial transfer technology will provide a new and effective treatment for infertility in aged women.


One of the important factors that affects female fertility is oocyte aging; decline in oocyte quality is the main cause of infertility in aged women. However, the main cause of oocyte aging and the molecular mechanism that regulates oocyte quality are still unclear. Previous studies have indicated that both the number and quality of mitochondria are involved in the quality control of oocytes and embryonic development1,2,3. The decrease in the quantity and quality of mitochondria is closely related to aging3.

Many attempts have been made to improve the function of mitochondria in aged oocytes, including the nutritional supplement of mitochondria and mitochondria transfer. Well-known, effective, nutritional supplements of mitochondria include Coenzyme Q10 (CoQ10), Alpha-lipoic acid (α-LA), and resveratrol (RSV)4. Studies have shown that CoQ10 supplementation can not only improve the age-related decline in the quantity and quality of oocytes, but also promote the normal development and ovulation of oocytes5. α-LA slows the oocyte quality decline related to aging and the metabolic phenotype of patients with polycystic ovary syndrome (PCOS)6,7. Resveratrol can reduce the number of oocytes with abnormal spindles and improper chromosome alignment increased in aging mice, while affecting the embryonic development in a dose-dependent manner8. However, as the clinical effect of nutritional supplements of mitochondria has not reached expected levels, other effective treatments need to be explored.

The first attempt of mitochondria transfer was carried out in 1997. The transfer of young donor oocyte cytoplasm into aged recipient oocytes improved the oocyte quality of the aged patients, who gave birth to healthy infants successfully9, which was the rationale behind the use of this technique. However, allogeneic oocyte cytoplasmic transfer cannot be applied to clinical practice due to two main reasons-the problem of genetic heterogeneity and regulatory issues caused by donor mitochondria transplantation. A previous study showed that autologous cell mitochondrial transplantation could improve the quality of oocytes, embryo development, and fertility of aged mice10, which had no ethical problems or genetic heterogeneity issues and solved some problems caused by the transfer of donor oocyte cytoplasm into recipient oocytes10,11.

Meanwhile, autologous cell mitochondrial transfer was superior to the effect of previous nutritional supplements of mitochondria on improving the quality of oocytes11. Therefore, autologous cell mitochondrial transplantation is the appropriate choice for the clinical application of this technology12. Adipose-derived stem cells (ADSCs) can be obtained by minimally invasive technology, are easy to isolate and culture, and can be an ideal "seed" cell for regenerative medicine. Mitochondria are rich in ADSCs, and the function of mitochondria does not decline with age, which suggests that ADSCs are an excellent source of mitochondria13,14. In this protocol, we introduce a method to transfer the mitochondria of mouse adipose-derived mesenchymal stem cells into aged mouse oocytes to improve the oocyte quality. This is a useful model for human ADSC autologous mitochondrial transfer technology.

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All the animal experiments described were approved by the Animal Research Ethics Committee of the Third Affiliated Hospital, Soochow University. All operations follow appropriate animal care and use agency and national guidelines. See the Table of Materials for details of all materials, instruments, and reagents used in this protocol.

1. Isolation and characterization of aged mouse adipose-derived mesenchymal stem cells (ADSCs)

  1. Sacrifice the aged mice (10-month-old, an average of three) by cervical dislocation under pentobarbital sodium anesthesia and soak them in 75% alcohol for 5 min before adipose tissue isolation.
    NOTE: Soaking in 75% alcohol for 5 min can effectively reduce the contamination of primary cells.
  2. Make a 2 cm incision in the skin on the bilateral inguinal, expose the subcutaneous fat, and use tweezers to isolate the subcutaneous fat. Collect the bilateral inguinal fat from the mice using sterilized ophthalmic scissors (avoid cutting the subcutaneous tissue). Place the adipose tissue in a 15 mL sterile centrifuge tube on ice and immediately transport it to the cell culture laboratory.
  3. Transfer the adipose tissue to a 6-well plate, rinse it 3x with phosphate-buffered saline (PBS) containing 100 U/mL of penicillin and streptomycin. Remove the blood vessels, fatty fascia, and connective tissue under the adipose tissue and cut them into ~0.5 cm x 0.5 cm x 0.5 cm pieces.
    NOTE: When isolating adipose stem cells, there is often contamination of vascular endothelial cells. Removal of the blood vessels from adipose tissue can reduce vascular endothelial cell contamination.
  4. Transfer the shredded adipose tissue to the same volume of collagenase type I solution (0.1% final concentration of collagenase type I) and digest at 37 °C for 30 min.
  5. Add an equal volume of complete culture medium (DMEM F-12 medium containing 10% fetal bovine serum) to neutralize the collagenase type I, centrifuge at 600 × g for 10 min, and discard the supernatant and adipose tissue.
  6. Resuspend the cell pellet in the complete culture medium. Remove the undigested tissue by filtration through a 40 µm cell strainer, and then centrifuge at 600 × g for 5 min.
  7. Resuspend the cell suspension with 5 mL of the complete culture medium and use a 1 mL pipette to gently pipette up and down to form a single-cell suspension. Check the cell suspension under a microscope by placing an aliquot on a hemocytometer to confirm that there are only singe cells, without any aggregates. Add the single-cell suspension to a 25 cm2 cell culture flask and culture in a 5% CO2 incubator at 37 °C.
  8. Change the culture medium 48 h later. Continue to change the culture medium every 2-3 days.
  9. After 7-10 days, detach the cells for cell passaging when their confluency reaches 80%-90%. Remove the complete culture medium and wash the cells with 2 mL of PBS. Then, add 2 mL of 0.05% trypsin/EDTA to perform the cell dissociation15,16.
  10. Add adipogenic induction medium for 14 days to induce differentiation of the ADSCs into adipoctyes. Add osteogenic induction medium for 28 days to the ADSCs plated on 2% gelatin coated 24-well plates to induce their differentiation into osteoblasts. For neural differentiation, culture the ADSCs with neural induction medium, then detect neuron-specific enolase and neurofilament mediator polypeptide using immunofluorescence15.
    1. Fix the cells in 4% paraformaldehyde for 30 min at room temperature, followed by permeabilization with 0.5% Triton X-100 in PBS for 15 min.
    2. After blocking with 3% BSA in PBS, incubate the cells with primary antibodies (see Table of Materials) diluted in blocking solution at 4 °C overnight.
    3. Incubate the cells with secondary antibodies for 45 min and then stain them with DAPI for 10 min. Then, examine the cells with a fluorescence microscope.
  11. Collect the single-cell ADSC suspension using 0.05% trypsin/EDTA, centrifuge at 600 × g for 5 min at room temperature, and resuspend in PBS to adjust the cell density to 1 × 106/mL. Aliquot the cell suspension into 1.5 mL microcentrifuge tubes (100 µL/tube) and add FITC-labeled rabbit anti-mouse CD29, CD90, CD34, and HLA-DR monoclonal antibodies, with an antibody concentration of 0.5 µg/mL. Treat the control group with the same volume of rabbit IgG FITC, incubate on ice for 30 min, and rinse with PBS to remove the unconjugated antibody. Detect the ADSC surface markers by flow cytometry13.

2. Isolation of mitochondria from adipose stem cells

NOTE: All the mitochondria isolation operations must be carried out on ice.

  1. When the ADSCs grow to 90% density, digest the cells with 2 mL of 0.05% trypsin/EDTA, centrifuge at 600 × g for 5 min, collect and count the cells, and take 1 × 107 cells for each extraction.
  2. Resuspend the cells in 2 mL of mitochondria extraction buffer (for buffer preparation: 10 mL of 0.1 M Tris-MOPS, 1 mL of 0.1 M EGTA/Tris, and 20 mL of 1 M sucrose; bring the volume to 100 mL with distilled water and adjust the pH to 7.4) with 1x protease inhibitor cocktail after washing with 2 mL of sterile PBS, and then incubate on ice for 5 min.
  3. Homogenize the cells 20x-30x with a glass homogenizer (2.0 mL) and check the degree of homogenization by trypan blue staining.
    NOTE: The proportion of the stained cells should be ~80%. Excessive homogenization will damage the structure of the mitochondria.
  4. Centrifuge the cell homogenate at 600 × g for 15 min, take the supernatant into a new, precooled 1.5 mL microcentrifuge tube, and repeat the operation once.
  5. Centrifuge the supernatant at 7,500 × g for 15 min and discard the supernatant. Wash the mitochondrial precipitate and resuspend it by adding ~100 µL of precooled mitochondrial injection buffer (225 mM mannitol, 75 mM sucrose, 10 mM KCl, 10 mM Tris-HCl, and 5 mM KH2PO4, pH 7.2), at a concentration of 1-5 mg/mL (total protein concentration). Transport the mitochondrial suspension on ice.
  6. Analyze the function and purity of the isolated mitochondria by JC-1 flow cytometric analysis and Western blot (VDAC1, β-actin, and Lamin B)13.
    1. Divide the isolated mitochondria into three groups: dimethyl sulfoxide (DMSO), JC-1 stained, and JC-1 stained with 30 min carbonyl cyanide 3-chlorophenylhydrazone (CCCP) pretreatment.
    2. After centrifuging at 7,500 × g, discard the supernatant and suspend the buffer.
    3. Capture the fluorescence intensity of the JC-1 monomers (FITC channel) and aggregates (PE channel) that were captured by flow cytometry. Evaluate the isolated mitochondrial membrane potentials by measuring the ratios of average fluorescence intensity of the PE channel to the FITC channel.
      ​NOTE: Perform the above steps in the dark.

3. Ovarian superstimulation

  1. Inject 10-month-old female C57BL/6 mice intraperitoneally with 10 IU of pregnant mare serum gonadotropin (PMSG) for superovulation. After 48 h, inject 10 IU of human chorionic gonadotropin (hCG) intraperitoneally.
  2. At 13 h after the injection of hCG, tear the swollen fallopian tube with microscopic tweezers. Release the cumulus complexes from the swollen fallopian tube. Dissolve the cumulus cells in M2 medium with hyaluronidase (0.3 mg/mL) at 37 °C.
    ​NOTE: The dissolution time is less than 5 min. Minimize the time for which the cumulus complex is exposed to hyaluronidase, as prolonged exposure can impair oocyte developmental potential.

4. Mitochondrial transfer along with ICSI

  1. Put the sperm without a tail in the mitochondrial liquid. Obtain sperm without a tail by cutting using ultrasound17. Place the sperm without a tail in the mitochondrial liquid, such that there are 100 sperm in 50 µL of the mitochondrial liquid.
    NOTE: As the mitochondrial suspension is slightly viscous and not stable in vitro, complete the injection within 30 min and change the microinjection needle immediately when it becomes clogged. The optimal inner diameter of the microinjection needle is 5 µm.
  2. Inject ~2 pL of mitochondrial respiration buffer or mitochondrial suspension with sperm into the oocytes using a microinjector under an inverted microscope (200x; see Table of Materials) within 30 min according to the protocol of Hiramoto as described by Mehlmann and Kline10,18.
    NOTE: The injected mitochondrial suspension accounts for 1%-3% of the total volume of oocytes. Control the volume (1%-3%) of the mitochondrial suspension in the cytoplasm to ensure the consistency of the injection.
  3. After injection, place the fertilized oocytes in M2 medium for equilibration for 15 min, and transfer the surviving fertilized eggs to M16 culture medium.
  4. Examine the surviving fertile eggs by observing the smooth morphology, pronuclear formation, and release of the second polar body19. Observe, photograph, and count the embryos at 09:00 a.m. and 06:00 p.m. every day for 4 days. Finally, collect and freeze the blastocysts and store them in a -80 °C refrigerator for ATP and mtDNA copy number determination to evaluate the number and improvement of mitochondrial transplantation.
    1. Prepare standard plasmid (plasmid for absolute quantification, as previously described13), for mitochondrial copy number determination according to the copy number of 1 × 107, 1 × 106, 1 × 105, 1 × 104, 1 × 103, 1 × 102, and 1 × 101. Transfer the embryos to the sterilization tube and add 20 µL of lysate buffer to release the mitochondrial DNA. Then, inactivate the protease in the lysate at 55 °C for 20 min and 95 °C for 10 min.
    2. Determine the mtDNA copy number by fluorescence quantitative PCR using the following setup: 5 µL of PCR mix, 0.5 µL of B6 primer-forward, 0.5 µL of B6 primer-rev, 2 µL of pyrolysis product, and 2 µL of ddH2O. Follow the PCR conditions: stage 1, 95 °C for 3 min; stage2, 95 °C for 30 s, 58 °C for 30 s, and 72 °C for 30s; repeat the cycle 40x.

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Representative Results

In this protocol, we isolated and characterized ADSCs from mouse fat (Figure 1). To obtain isolated mitochondria, the cell membrane must be disrupted using a glass homogenizer. (Figure 2A). It is important to obtain a uniform mitochondrial fraction without large clumps so that the microinjection tube is not blocked. First, 200 µL, and then 10 µL, pipette tips must be used to resuspend the homogenates gently; finally, a 29 G needle must be used to slowly aspirate two or three times to obtain the mitochondrial components that can be used for injection (Figure 2B). Mitochondrial membrane potential fluorescent probes were used for labeling to ensure that the extracted mitochondria were active (Figure 2C). The purity and function of mitochondria were analyzed by JC-1 flow cytometry and Western blot (Figure 2D,E).

To minimize the microinjection time, sperms with the tail removed were placed in the mitochondrial fraction beforehand, and the mitochondrial fraction was equilibrated from frozen to warm to room temperature during injection (Figure 3A,B). In vitro embryo experiments are required to assess whether oocyte quality is improved after mitochondrial transfer. Embryonic development must be monitored every day, and the embryonic development rate counted to determine whether the mitochondrial transfer is effective (Figure 3C). We found the blastocyst formation rate of mice receiving mitochondrial transfer was improved compared with the rate of mice in the control group (Figure 3D).

Figure 1
Figure 1: Isolation and characterization of aged mouse adipose-derived mesenchymal stem cells. (A) Isolation of bilateral inguinal fat. (B,C) Representative images of trimmed adipose tissue. (D) Adipogenic differentiation of ADSCs; the formation of lipid droplets was confirmed by Oil Red O staining. (E) Osteogenic differentiation of ADSCs; calcium deposition was observed.(F,G) Differentiation of ADSCs to neuronal lineage was confirmed by immunofluorescence staining of NSE and NFM. (H-K) The flow cytometry analysis of ADSC surface markers.ADSCs are positive for CD29 and CD90 but negative for CD34 and HLA-DR. Scale bars = 100 µm.Abbreviation: ADSCs = adipose-derived stem cells; NFM = neurofilament medium polypeptide; NSE = neuron-specific enolase; FITC-A = fluorescein isothiocyanate peak area. Please click here to view a larger version of this figure.

Figure 2
Figure 2: Mitochondria isolation from ADSCs. (A) ;Glass homogenizer. (B) Representative images of isolated mitochondria. (C) Representative images of isolated mitochondria stained by MitoTracker Red. (D) The function of mitochondria was analyzed by JC-1 flow cytometry. (E) Proteins of homogenate, supernatant, and isolated mitochondria fractions were compared by Western blot analysis. An anti-β-actin antibody was used to detect cytoplasmic marker proteins, an anti-Lamin B antibody was used to detect nuclear protein, and an anti-VDAC1 antibody was used to detect mitochondrial marker protein. Scale bars = 500 µm (5x) (A), 50 µm (40x) (B), 100 µm (C). Abbreviations: ADSCs = adipose-derived stem cells; BF = brightfield; DMSO = dimethyl sulfoxide; CCCP = carbonyl cyanide 3-chlorophenylhydrazone. Please click here to view a larger version of this figure.

Figure 3
Figure 3: Mitochondria transfer of ADSCs into an aged mouse oocyte. (A) Image of sperm head in isolated mitochondria during transfer. (B) Images of mitochondria transfer. (C) Images of blastocyst formation after mitochondrial transfer/buffer control transfer. The images on the right are zoomed-in images of the dashed rectangles on the left images. (D) The blastocyst formation rate of mice receiving mitochondrial transfer compared with the rate of mice in the control group. Scale bars = 50 µm. (B,C). Abbreviations: ADSCs = adipose-derived stem cells; Ctrl = control; MIT = mitochondrial transfer. Please click here to view a larger version of this figure.

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Oocytes contain more mitochondria than any type of cell in the body, with ~1-5 × 105 mtDNA copy numbers. Mitochondria are essential for oocyte maturation, fertilization, and embryonic development, thus, any mitochondrial dysfunction can lead to decreased oocyte quality. Decreased mitochondrial quantity and quality are closely related to physiological aging. In this protocol, a simple method for isolating mitochondria from the ADSCs of aged mice and transfer to aged mouse oocytes was introduced to attempt to improve aged oocyte quality.

Since scientists conducted the world's first oocyte cytoplasmic transplantation experiment in 1997 and successfully improved the quality of oocytes in elderly patients, the mitochondrial transfer technology developed has been the focus of much attention. However, the allogeneic mitochondrial transfer raises ethical, legal, and potential long-term health problems of mitochondrial genetic heterogeneity. In recent years, due to the discovery of oogonial stem cells, autologous oogonial stem cell mitochondrial transplantation has attracted more attention; however, oogonial stem cell acquisition requires a highly invasive method and is difficult to culture and purify, which limits its applicability20. ADSCs can be obtained using minimally invasive techniques and are easy to isolate and culture21. They are ideal "seed" cells for regenerative medicine. More importantly, studies have shown that the stemness and proliferative capacity of ADSCs do not decline with age22, suggesting that they may be ideal candidates for autologous mitochondrial transfer. Thus, in this protocol, we chose ADSCs from an aged mouse as a model for human ADSC autologous mitochondrial transfer to provide a basis for animal studies for clinical application.

The following mitochondrial separation and purification methods are commonly used23: i) sucrose gradient or Percoll ultracentrifugation based on the gradient centrifugation (GC) method; ii) differential centrifugation (DC)-simple and fast but difficult to obtain pure mitochondria; and iii) free-flow electrophoresis (FEE), which requires special equipment (ProTeam FFE apparatus) and special expertise. By comparing the traditional GC/FEE/commercial kit methods, some studies have concluded that the highest mitochondrial purity obtained is nearly 70% by the FEE method, 57% by the GC method, and 50% by the DC method. Considering the ease of operation and the yield, purity, integrity, and functionality of mitochondria obtained, the Differential Density Centrifugation Strategy has a higher prospect and possibility. The isolated mitochondria in vitro are very fragile, while the GC and FEE methods cost hours to obtain higher mitochondria purity, which also affects the quality of the mitochondria. However, the DC method can obtain mitochondria within 50 min and preserve mitochondrial integrity and function. In this protocol, we introduced a modified DC method that preserved mitochondria purity and function.

Mitochondrial transfer was the most important step to get the best results. During the mitochondrial transfer process, we found that the mitochondrial fraction was viscous and would often clog the microinjection tube. Hence, we suggest that the mitochondrial transfer process should be completed as soon as possible to avoid impairment of the developmental potential of oocytes. One of the biggest challenges is accurately measuring the number of mitochondria being injected and determining how many of them will survive and function, a focus of future research. In conclusion, mitochondrial transfer of autologous adipose-derived stem cells is a promising technology, which may effectively improve the quality of oocytes caused by aging. However, there are still some technical difficulties that need to be further solved by follow-up research.

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The authors have no conflicts of interest to disclose.


The authors wish to acknowledge support from the National Nature Science Foundation of China (82001629 to X.Q.S.), the Basic Research Project of Changzhou science and Technology Bureau under grant number CJ20200110 (to Y.J.Y.), the Youth Program of Natural Science Foundation of Jiangsu Province (BK20200116 to X.Q.S.), and Jiangsu Province Postdoctoral Research Funding (2021K277B to X,Q.S.).


Name Company Catalog Number Comments
0.05% trypsin/EDTA Gibco 25300054 Cell Culture
 4% paraformaldehyde beyotime P0099 immunofluorescence
40 μm cell strainer Corning 352340 ADSC isolation
adipogenic induction Cyagen HUXXC-90031 Multidirectional differentiation
Alizarin red staining solution Sigma A5533 Multidirectional differentiation
Antibody against CD29 BD Biosciences 558741 flow analysis
Antibody against CD34 BD Biosciences 560942 flow analysis
Antibody against CD90 BD Biosciences 553016 flow analysis
Antibody against HLA-DR BD Biosciences 555560 flow analysis
β-actin Abcam ab-8226 Mitochondrial function test
BSA Sigma V900933 immunofluorescence
CCCP Solarbio C6700 mitochondria JC-1 flow analysis
ChamQ Universal SYBR qPCR Master Mix Vazyme Q711 qPCR
collagenase type I Sigma SCR103 ADSC isolation
DAPI  Invitrogen D1306 immunofluorescence
DMEM-F12 Gibco 11320033 Cell Culture
DMSO Sigma 276855 mitochondria JC-1 flow analysis
EGTA Sigma 324626 Mitochondria isolation
FBS Gibco 10100147 Cell Culture
Flow cytometry BD Biosciences FACSCanto™ II Characteristics of ADSCs
fluorescence microscope leica DM2500 immunofluorescence
gelatin Sigma 48722 Multidirectional differentiation
glass homogenization tube Sangon F519062 Mitochondria isolation
hCG Aibei M2520 Ovarian superstimulation
hyaluronidase Sigma H1115000 Ovarian superstimulation
 Inverted microscope Olympus IMT-2 Microinjection
Isolated Mitochondria Staining Kit Sigma CS0760 mitochondria JC-1 flow analysis
JC-1 Sigma T4069 Mitochondrial function test
KCl Sigma P5405 Mitochondria transfer
KH2PO4 Sigma P5655 Mitochondria transfer
LaminB Abcam ab-16048 Mitochondrial function test
M16 Medium Sigma M7292 embryo cell culture
M2 Medium Sigma M7167 embryo cell culture
mannitol Sigma M9546 Mitochondria transfer
Microinjector Olympus+ eppendorf IX73 Mitochondria transfer
MitoTracker red Invitrogen M22425 Mitochondria staining
MOPS Sigma M1442 Mitochondria isolation
neurofilament mediator polypeptide (NFM) Santa Cruz Biotechnology sc-16143 Multidirectional differentiation
neurogenic induction Gibco A1647801 Multidirectional differentiation
Neuron-specific enolase (NSE) Santa Cruz Biotechnology sc-292097 Multidirectional differentiation
Oil Red O Sangon E607319 Adipogenic differentiation
oil red O solution Sigma O1516 Multidirectional differentiation
osteogenic induction Cyagen HUXXC-90021 Multidirectional differentiation
PBS (phosphate buffered saline) Hyclone SH30256.LS Cell Culture
penicillin and streptomycin Hyclone SV30010 Cell Culture
PMSG Aibei M2620 Ovarian superstimulation
protease Inhibitor cocktail Sigma P8340 Mitochondria isolation
sucrose Sigma V900116 Mitochondria isolation
Tris Sigma 648314 Mitochondria isolation
Tris-HCl Sigma 108319 Mitochondria transfer
Triton X-100 beyotime P0096 immunofluorescence
VDAC Abcam ab-14734 Mitochondrial function test



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Cite this Article

Yang, Y., Zhang, C., Sheng, X. Mitochondrial Transfer from Mouse Adipose-Derived Mesenchymal Stem Cells into Aged Mouse Oocytes. J. Vis. Exp. (191), e64217, doi:10.3791/64217 (2023).More

Yang, Y., Zhang, C., Sheng, X. Mitochondrial Transfer from Mouse Adipose-Derived Mesenchymal Stem Cells into Aged Mouse Oocytes. J. Vis. Exp. (191), e64217, doi:10.3791/64217 (2023).

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