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

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 dissociation^15,16.
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 Kline^10,18.
   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.