Isolation and Identification of Porcine Bone Marrow Mesenchymal Stem Cells and their Derived Extracellular Vesicles

According to the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health, USA, all the experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC), Fuwai Hospital, Chinese Academy of Medical Sciences.

1. Preoperative preparation for the animals

1. Obtain adult male Chinese minipigs (30 ± 5 kg) at about 12 months old from the Institute of Zoology, Chinese Academy of Sciences, and house them in the facilities of Animal Experimental Center, Fuwai Hospital, at least 2 weeks in advance. Carry out preoperative inspections such as blood routine examinations to ensure that the animals are healthy.
2. Clean and shave the skin of the thigh area of the minipig the day before the operation. To avoid aspiration, fast the minipig for 12 h before bone marrow extraction.

2. Preparation for cell isolation and cultivation

1. Use Percoll (1.130 g/mL) to isolate mesenchymal stem cells from bone marrow. Mix the stock solution with 10x concentrated PBS at a ratio of 9:1 to obtain an isotonic medium. Then mix the isotonic medium with PBS at a ratio of 3:2 to acquire 60% Percoll solution (1.077 g/mL), which can be used as the final separating solution to isolate pBM-MSCs.
2. Prepare Iscove's Modified Dulbecco's Medium (IMDM) with 10% fetal bovine serum and 1.0% penicillin-streptomycin to obtain a complete medium. Preheat the complete medium and PBS in a 37 °C water bath for subsequent cell cultivation.

3. Anesthetization for the animals

1. Administer general anesthesia with ketamine (10 mg/kg) and xylazine (2 mg/kg) intramuscularly. Carry out endotracheal intubation quickly when the minipig has slow respiration and fewer limb activity, and preserve spontaneous breathing to prevent failed intubation.
2. Perform maintenance of anesthesia by inhalation of 2% isoflurane, with oxygen (1.5 L/min) as the carrier gas. During anesthesia, monitor heart rate, respiration, and blood oxygen saturation of the minipig in real-time.

4. Extracting bone marrow from the minipig

1. Place the minipig in a lateral position. To ensure a sufficient amount of bone marrow for subsequent cell culture, locate the bone marrow puncture point at the proximal femur of the minipig (Figure 1). Disinfect and drape the skin of the puncture area before the operation is applied.
2. Tighten the skin around the puncture point with the non-dominant hand, and pierce the bone marrow biopsy needle vertically at the point with the dominant hand. When feeling the needle in contact with the cortical bone, gently rotate the handle left and right to drill the needle. When the needle enters the bone marrow cavity, there is often a feeling of losing resistance.
3. After the puncture is in place, withdraw the core needle. Then attach a disposable sterile 50 mL syringe to the end of the outer needle, and rinse the inner wall of the syringe with heparin in advance.
4. Extract 20 mL of bone marrow slowly and transfer them to a sterile 50 mL centrifuge tube carefully.
5. When the procedure is complete, pull out the needle and remove the drape. Disinfect the puncture site and press it for 20 min for hemostasis.
6. Extubate the minipig after restoring spontaneous breathing. When it is fully awake with free movement of limbs, return the minipig to the cage to continue feeding.

5. Isolating mesenchymal stem cells from the bone marrow

1. Add an equal volume of preheated PBS to the bone marrow and mix them thoroughly. Then use a sterile pipette to transfer 20 mL of the diluted bone marrow above the 60% density gradient solution level at a 1:1 volume ratio in a sterile 50 mL centrifuge tube carefully.
2. Centrifuge the tube at 600 x g ( acceleration (ACC) = 5, deceleration (DEC) = 5) for 20 min at room temperature (RT).
   NOTE: Four phases are formed in the tube after centrifugation, including the serum phase, mononuclear cell phase, density gradient medium phase, and precipitation phase from top to bottom. The mononuclear cell phase is a thin flocculent layer between the serum and the density gradient medium. Mesenchymal stem cells derived from the bone marrow are located in this phase.
3. Draw the mononuclear cell phase into a sterile 15 mL centrifuge tube, and wash twice with PBS at 800 x g for 5 min.
4. Resuspend the washed cells in 2 mL of complete medium and plant the resuspended cells in a 175 cm² cell culture flask at a density of 3-5 x 10⁵/mL.
   ​NOTE: All the above procedures for isolating mesenchymal stem cells can be seen in Figure 2.

6. Cultivating mesenchymal stem cells in vitro

1. Incubate the culture flask at 37 °C in a saturated humidified atmosphere of 5% CO₂. Shake the culture flask gently every 24 h to prevent adherent growth of the precipitated hematopoietic stem cells, and observe cell growth, morphology, and contamination under a microscope.
2. Replace the culture medium for the first time after 3 days, and then change the medium every 2-3 days. When the cell colonies reach 80%-90% confluence, subculture the cells at a ratio of 1:2.

7. Adipogenic, osteogenic, and chondrogenic differentiation of pBM-MSCs

1. Adipogenic differentiation assay
   1. Prepare adipogenic differentiation medium A (Medium A) and B (Medium B) for BM-MSCs according to the kit instructions. Specific information about the kit can be found in the Table of Materials.
   2. Add 1 mL of 0.1% gelatin to the six-well plate and shake gently so that it can cover the bottom of each well evenly. Then place the six-well plate in a clean bench or CO₂ incubator for at least 30 min.
   3. After 30 min, aspirate the gelatin and add 2 mL of general complete medium to each well. Then plant the pBM-MSCs in the six-well plate at a cell density of 2 x 10⁴ cells/cm². After that, incubate the plate at 37 °C in saturated humidity of 5% CO₂.
   4. When the cells reach 100% confluence, remove the complete medium carefully and add 2 mL of Medium A to each well of the plate. After 3 days, remove Medium A from the plate and add 2 mL of Medium B to each well.
   5. After maintaining for 1 day, remove Medium B and replace it with Medium A for induction. According to the manner of "Medium A for 3 days, Medium B for 1 day", use Medium A and B sequentially for induction.
   6. Observe the cell state every day during the period. If the cells shrink or die during the induction process of Medium A, replace it with Medium B in time until the cell state recovers.
   7. Repeat the induction and maintenance process, and prepare for staining when sufficient lipid droplets of suitable size are observed under the microscope.
   8. Remove the medium for adipogenic differentiation in the six-well plate, and wash with 1x PBS gently. Add 2 mL of 4% paraformaldehyde solution to each well and fix for 30 min at RT.
   9. Remove the paraformaldehyde fixative and wash with 1x PBS two or three times to ensure that the fixative is removed thoroughly. Add 2 mL of Oil Red O dye to each well and stain for 30 min at RT.
   10. Remove the Oil Red O dye and wash with 1x PBS two or three times. Add 2 mL of 1x PBS to each well, and then observe the effect of adipogenic differentiation under a microscope.
2. Osteogenic differentiation assay
   1. Prepare the complete medium for osteogenic differentiation according to the kit instructions.
   2. Follow steps 7.1.2-7.1.3 to plant and culture pBM-MSCs.
   3. When the cells reach 70% confluence, remove the general complete medium carefully and add 2 mL of medium for osteogenic differentiation to each well of the plate.
   4. Change to fresh osteogenic differentiation medium every 3 days. Continue the induction for 2-4 weeks, and prepare to stain with Alizarin Red dye when obvious calcium nodules appear during osteogenesis.
   5. Remove the medium for osteogenic differentiation in the six-well plate, and wash with 1x PBS gently. Add 2 mL of 4% paraformaldehyde solution to each well and fix for 30 min at RT.
   6. Remove the paraformaldehyde fixative and wash with 1x PBS two or three times to ensure that the fixative is cleaned thoroughly. Add 2 mL of Alizarin Red dye to each well and stain for 10 min at RT.
   7. Remove the Alizarin Red dye and wash with 1x PBS two or three times. Add 2 mL of 1x PBS to each well, and then observe the effect of osteogenic differentiation under a microscope.
3. Chondrogenic differentiation assay
   1. Prepare the premix for chondrogenic differentiation according to the kit instructions.
   2. Transfer 3-4 x 10⁵ pBM-MSCs to a sterile 15 mL centrifuge tube. Centrifuge at 250 x g for 4 min at 20 °C.
   3. Remove the supernatant and add 0.5 mL of premix to resuspend the pellet obtained by centrifugation in the previous step, and then centrifuge at 150 x g for 5 min at 20 °C. Repeat this step to wash the cells again.
   4. Prepare the complete medium for chondrogenic differentiation.
   5. Resuspend the cell obtained in the previous step with 0.5 mL of complete medium and centrifuge at 150 x g for 5 min at 20 °C.
   6. Unscrew the cap of the centrifuge tube to facilitate gas exchange. Place it upright in an incubator at 37 °C, 5% CO₂, and saturated humidity.
   7. When the cells appear to aggregate (usually after 24-48 h, depending on the actual situation), flick the bottom of the centrifuge tube to make the cartilage balls detach from the bottom and suspend in the medium.
   8. Change to fresh complete medium for chondrogenic differentiation every 2-3 days. Continue the induction until cartilage balls with a diameter of 1.5-2 mm are formed in the tube, and then prepare sections for staining.
   9. Prepare paraffin sections of cartilage balls according to the routine steps of pathological experiments. Add Alicia blue dye to the de-waxed sections and stain at 37 °C for 1 h.
   10. Rinse the slide with running water for 5 min, and then observe the staining effect of Alicia blue under a microscope after drying.

8. Identification of cell phenotype by flow cytometry

1. When the cell colonies reach 80%-90% confluence at passages 3-5, remove the culture medium and wash the cells twice with preheated PBS. Then digest the cells with 3-4 mL of 0.25% trypsin/EDTA and incubate them at 37 °C in a saturated humidified atmosphere of 5% CO₂ for 2-3 min until they are detached from the bottom of the flask under a microscope.
2. Harvest the cells with a 10 mL complete medium and transfer the cell suspension into a sterile 15 mL centrifuge tube. Centrifuge the cell suspension at 800 x g for 5 min at RT. Discard the supernatant and wash the cells with 4 °C PBS.
3. Resuspend the cells to 10 mL with 4 °C PBS. The cell suspension is divided into nine groups with a volume of 1 mL in each 1.5 mL microtube, named negative control, FITC isotype control, PE isotype control, APC isotype control, CD105, CD29, CD90, CD14, and CD45 group, respectively. Ensure that the number of cells in each microtube is between 1 x 10⁵ and 1 x 10⁶.
4. Centrifuge the suspension at 800 x g for 5 min at 4 °C, and resuspend the cells in each microtube with 100 µL of 4 °C PBS again. Except for the negative control group, add 5 µL of the corresponding isotype control (FITC, PE, and APC Mouse IgG1 kappa Isotype Control) and antibodies (CD105, CD29, CD90, CD14, and CD45 Monoclonal Antibody) for flow cytometry to each microtube according to the order in step 8.3. Mix gently and incubate for 1.5 h at 4 °C in the dark.
5. Add 1 mL of 4 °C PBS to each microtube and centrifuge at 300 x g for 10 min at 4 °C. Discard the supernatant and add 200 µL of 4 °C PBS to resuspend the cells.
6. Test at least 10,000 cells on the flow cytometry after filtering the cell suspension¹⁶ and analyze the data using the flow cytometry software.

9. Isolating extracellular vesicles (EVs) derived from porcine bone marrow mesenchymal stem cells

1. When the pBM-MSCs confluence reaches 80%-90%, discard the supernatant and wash cells with PBS 2x. Then add 25 mL of serum-free IMDM to each culture flask and continue to incubate at 37 °C in a humidified atmosphere of 5% CO₂ for 48 h.
2. Collect the cell supernatant (conditioned medium, CM) into a 50 mL centrifuge tube and centrifuge at 300 x g for 10 min at 4 °C to remove the cell debris.
3. Collect the supernatant again into another 50 mL centrifuge tube. Carry out the isolation of EVs as soon as possible after collecting the supernatant. For long-term storage, store the supernatant in the refrigerator at -80 °C to prevent the loss of EVs.
4. Centrifuge the supernatant in step 9.3 at 2,000 x g for 20 min at 4 °C.
5. Transfer the supernatant to a sterile tube used for high-speed centrifuge and centrifuge at 16,500 x g for 30 min at 4 °C.
6. Transfer the supernatant again to an ultracentrifuge tube, and centrifuge at 120,000 x g for at least 70 min at 4 °C, with a fixed angle rotor.
7. Discard the supernatant completely. Add 1 mL of 4 °C PBS to each ultracentrifuge tube and resuspend the precipitation with a micropipette. Mix the solution from the same group into an ultracentrifuge tube, and then add 4 °C PBS to make the volume more than 3/4 of the tube.
8. Centrifuge at 120,000 x g for 60 min at 4 °C and remove the supernatant as much as possible.
9. Resuspend the precipitant with sterile PBS again, and store the EVs in a -80 °C refrigerator.
   NOTE: The amount of PBS for resuspending can be determined as 100 µL for the precipitation from every two 175 cm² culture flasks. All above steps of isolating EVs are shown in Figure 3 systematically.

10. Identification of EVs by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and western blotting

1. Place the EVs sample on ice after thawing it in a 25 °C water bath, and then dilute to half of the previous concentration with PBS for NTA detection¹⁷.
2. Pipette 10 µL of the sample on the copper mesh, allow it to settle for 1 min, and absorb the floating liquid with filter paper. Then, add 10 µL of phosphotungstic acid to the copper mesh, allow it to stand for 1 min, and absorb the excess liquid.
3. After drying for a few minutes at RT, perform electron microscopy imaging under the condition of 100 KV accelerating voltage¹⁸.
4. Resuspend the EVs sample from step 9.9 in RIPA lysis buffer (25 mM Tris·HCl (pH 7.6), 150 mM sodium chloride [NaCl], 1% nonyl phenoxypolyethoxylethanol (NP-40), 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 1 mM Phenylmethanesulfonyl fluoride (PMSF), 1x protease inhibitor), and detect the expression of specific markers for EVs such as Alix, TSG101, CD81, and CD63 by western blotting¹⁹.