NOTE: The use of human fibroblasts may require ethical approval. Fibroblasts used in this study were derived from MD patients and stored in the Institutional biobank in compliance with ethical requirements. Informed consent was provided for the use of the cells. Perform all cell culture procedures under a sterile laminar flow cabinet at room temperature (RT, 22-25 °C). Use sterile-filtered solutions suitable for cell culture and sterile equipment. Grow all cell lines in a humidified incubator at 37 °C with 5% CO₂. Mycoplasma tests should be conducted weekly to ensure mycoplasma-free cultures. 143BTK^– rho⁰ cells can be generated as previously described⁵.

1. Culture of cells

1. Before starting any procedure, verify the presence of the mutation in fibroblasts derived from MD patient(s) and quantify the percentage of heteroplasmy or homoplasmy by Restriction Fragment Length Polymorphism (RFLP) and/or whole mtDNA sequencing analyses¹⁴.
2. Seed fibroblasts in four 35 mm Petri dishes, each containing 2 mL of Complete Culture Medium (Table 1). Let the cells grow until 80% confluent (48 h).
3. Grow 143BTK^– rho⁰ cells in 8 mL of Supplemented Culture Medium (Table 1) in a 100 mm Petri dish.
4. Maintain the cells in an incubator at 37 °C with 5% CO₂.
5. Check the absence of mtDNA in rho⁰ cells by sequencing techniques¹⁴.

2. Enucleation of fibroblasts

1. Sterilize four 250 mL centrifuge-suitable bottles by autoclave sterilization at 121 °C for a 20 min cycle. Dry them in a laboratory oven or at RT.
2. Prewarm the centrifuge at 37 °C.
3. Wash the 35 mm dishes containing the fibroblasts twice, using 2 mL of 1x phosphate-buffered saline (PBS) without (w/o) calcium and magnesium.
4. Clean the outer surface of the dishes with 70% ethanol and wait until the alcohol evaporates.
5. Remove the lids from the dishes and the screw caps from the bottles. Place each dish, without the lid, upside down on the bottom of each 250 mL centrifuge bottle.
6. Slowly add 32 mL of Enucleation Medium to each bottle (Table 1), allowing the medium to enter the dish and come into contact with the cells. Remove any bubbles from the dishes using a long glass Pasteur pipette, curving the tip in a Bunsen flame.
   NOTE: It is important to remove the bubbles to allow the medium to enter the dish and come in contact with the cells.
7. Close each bottle with the screw cap and transfer them to the centrifuge.
8. Centrifuge for 20 min at 37 °C and 8,000 × g, acceleration max, deceleration slow. Pay attention to balance the centrifuge: counterweight each bottle. If necessary, adjust the weight by adding a suitable volume of Enucleation Medium.
9. During centrifugation, use vacuum or a 10 mL serological pipette to aspirate and discard the medium from the 143BTK^– rho⁰ culture plates and wash them twice using 4 mL of 1x PBS w/o calcium and magnesium.
10. Add 2 mL of trypsin to cover the cell monolayer completely.
11. Place the dishes in a 37 °C incubator for ~2 min.
12. Remove the dishes from the incubator, observe cell detachment using an inverted microscope for live cells (objectives 4x or 10x), and inhibit the enzyme activity by adding 2 mL of Supplemented Culture Medium.
13. Aspirate the 4 mL of the cell suspension in the dish with a 10 mL pipette and transfer it to a 15 mL conical tube.
14. Count the cells using a Burker hemocytometer chamber or an automated counter.
15. At the end of centrifugation (step 2.8), aspirate the medium from the bottles and discard it.
16. Remove the dishes by inverting the bottles on a sterile gauze previously sprayed with 70% ethanol. Clean the outer surface of the dishes and their lids with 70% ethanol. Wait until the alcohol evaporates, and then close the dishes.
17. Before proceeding, check for cytoplast (ghost) formation using an inverted microscope for live cells (objective 4x or 10x). Look for extremely elongated fibroblasts due to the extrusion of their nuclei induced by cytochalasin B.
18. To each 35 mm dish, add 1 × 10⁶ of 143BTK^– rho⁰ cells resuspended in 2 mL of 143BTK^– rho⁰ culture medium supplemented with 5% fetal bovine serum (FBS).
19. Leave the dishes for 3 h in a humidified incubator at 37 °C and 5% CO₂ and let the 143BTK^– rho⁰ cells settle on the ghosts. Do not disturb the dishes.

3. Fusion of the enucleated fibroblasts with rho ⁰ cells

1. After 3 h of incubation, aspirate and discard the medium from the dishes.
2. Wash the adherent cells twice with 2 mL of Dulbecco's Modified Eagle Medium (DMEM) high glucose w/o serum or with Minimum Essential Medium (MEM).
3. Aspirate and discard the medium.
4. Add 500 µL of PEG solution (see the Table of Materials) to the cells and incubate for exactly 1 min.
5. Aspirate and discard the PEG solution.
6. Wash the cells three times using 2 mL of DMEM high glucose w/o serum or with MEM.
7. Add 2 mL of Fusion Medium (Table 1) and incubate overnight in the incubator at 37 °C with 5% CO₂.

4. Cybrid selection and expansion

1. After overnight incubation, remove the plates from the incubator, trypsinize the cells as described above (steps 2.9-2.13), and transfer the content of each 35 mm dish into a 100 mm dish.
2. Add 8 mL of Selection Medium (Table 1) and place the plates in the incubator at 37 °C with 5% CO₂.
3. Change the medium every 2-3 days.
4. Wait for ~10-15 days of selection until colonies of cells appear.
5. Freeze one of the four Petri dishes by collecting all the clones and generating a "massive" culture as a backup of the cybrids, which can be eventually recloned and used for further investigations.
6. Trypsinize the cells in the remaining culture dishes, count, and seed into one or more Petri dishes at 50-100 cells/dish in the Supplemented Culture Medium (Table 1) until clones appear. Let them grow for some days.
7. Pellet the remaining cells by centrifugation at 1,200 × g for 3 min at RT and discard the supernatant.
8. Extract DNA from the pellet (see the Table of Materials).
9. Perform genotyping by variable number of tandemly repeated (VNTR) analysis as previously reported¹⁵.
10. Pick up clones from the Petri dish with cloning cylinders or a pipette tip, using a stereomicroscope to avoid pooling of different clones, and transfer them to a 96-well plate, each well containing 200 µL of Supplemented Culture Medium (Table 1).
11. Expand every clone until there are enough cells for freezing and extracting DNA.
12. Verify the mutation percentage of each clone by RFLP or other sequencing methods. Ideally, try to obtain both clones with wild-type mtDNA (0% mutation) and clones with different mutation percentages, both adding up to homoplasmic mutant mtDNA (100% mutation). See Figure 1 for a schematic diagram of the cybrid generation protocol.