Generating Plasma Membrane Vesicles from Bone Marrow Stem Cells

1. Assembly of the Apparatus

1. To ensure sterility, turn on the ultraviolet (UV) light of a tissue culture hood for 30 min before use.
2. Unscrew a disposable 25 mm filter unit and submerge the cap and bottom of the unit into a 200 mL glass cup filled with 75% ethanol for 30 min. The unit is made of medical-grade polypropylene.
3. Pick up the cap and bottom of the unit using forceps, shake off the remaining ethanol by hand, and allow the parts to air dry for 10 min in the hood.
4. Wet a 19 mm polycarbonate membrane in phosphate-buffered saline (PBS) and place it carefully on top of the supporting matrix of the bottom of the unit. The polymer film has a smooth, flat surface and track-etched 3 µm pores.
5. Reassemble the unit by screwing the cap tightly against the bottom. Make sure that the membrane is not dislodged from the center.
6. Remove the needle of a 1 mL insulin syringe and draw 1 mL of PBS. Attach the syringe to the filter unit and then push PBS through the unit to wet the assembly and to test if there is any leakage.

2. Generation of Plasma Membrane Vesicles (PMVs)

1. Establish a bone marrow mesenchymal stem cell (BMSC) culture, as reported by Nemeth et al., in Dulbecco's Modified Eagle Medium (DMEM) culture medium containing 15% fetal bovine serum (FBS) and 1% penicillin/streptomycin. Cultivate the cells in a 6-well plate in a humidified incubator containing 5% CO₂ at 37 °C.
2. To propagate the cells, remove the culture medium and rinse the well with 0.5 mL of calcium-free PBS.
3. Add 0.5 mL of 0.25% trypsin-ethylenediamine tetraacetic acid (trypsin-EDTA) and incubate the cells at 37 °C for 3 min. Tap the plate against the palm of the hand a couple of times to facilitate cell detachment. Stop digestion by adding 1 mL of culture medium.
4. Collect the cells in a 15 mL conical tube and spin at 200 x g for 2 min in a bench-top centrifuge. Resuspend the cells in 4 mL of culture medium and aliquot the cells into two wells of a 6-well plate.    
   NOTE: Cells usually reach confluence at around 24 h.
5. To generate PMVs, harvest the cells from one well (about 5 x 10⁵ cells) of a 6-well plate by trypsin digestion and monodisperse the cells in 0.5 mL of culture medium.
6. Load the cells into the insulin syringe, attach it to the filter unit, and push the plunger quickly to squeeze the cells through the filter. Discard the extruded medium because there should only be a few PMVs inside it.
7. Load an additional 0.5 mL of medium into the syringe and quickly push it through the filter. Collected the medium of the second extrusion, which contains PMVs of various sizes.
8. Load 150 µL of the collected medium into a 35-mm glass-bottom dish and allow the PMVs to settle to the bottom for about 10 min. Examine the PMVs under an inverted phase contrast microscope equipped with a CCD camera using the 20X objective lens.