To begin, collect the supernatant from the human adipose-derived mesenchymal stem cell culture once the cells are 80%confluent. Collect the supernatant carefully using a pipette and transfer it to a 50 milliliter centrifuge tube. Centrifuge the tube at 300 x g for 10 minutes at room temperature to remove suspended cells, then use a pipette to collect the supernatant without disturbing the pellet.
Centrifuge the supernatant at 2000 x g for 10 minutes at four degrees Celsius to remove cellular debris. Collect the resultant supernatant with a pipette and filter it through a 0.22 micron membrane to remove large particles and cell debris. Subject the filtrate to ultra centrifugation at 10, 000 x g for 30 minutes at four degrees Celsius.
Then perform a final centrifugation at 100, 000 x g for 70 minutes at four degrees Celsius. Using a pipette, remove the supernatant without collecting the pellet. Resuspend the pellet in PBS before centrifuging the tube at 100, 000 x g for 70 minutes at four degrees Celsius.
Remove the supernatant and resuspend the obtained pellet of extracellular vesicles, or EVs, in one milliliter of PBS. Finally, transfer the EV suspension to a one milliliter centrifuge tube and store it at four degrees Celsius until further analysis. Transmission electron microscopy clearly revealed the cup-shaped structure of the EVs with a bilayer membrane.
Nanoparticle tracking analysis indicated that they have a particle size distribution of around 50 to 150 nanometers. Western blot analysis showed that the characteristic marker proteins of EVs were expressed smoothly.
