Our research focuses on the preparation and purification of extracellular vesicles from human adipose derived mesenchymal stem cells. The problem we want to solve is how to standardize this process and improve the purification efficiency. Currently, there are many technologies used to extract extracellular vesicles, such as ultracentrifugation, ultra filtration, volume exclusion chromatography, polymer precipitation, immunoaffinity capture, microfluidic technology, and so on.

However, ultracentrifugation is recognized as a gold standard, admittedly. In the future, we will focus on the application of extracellular vesicles as vectors, including carrying mRNA, plasmids, and other small molecules and transfecting them into cells. To begin, retrieve two cryo-preservation tubes containing frozen human adipose-derived mesenchymal stem cells at passage three from liquid nitrogen.

Agitate them in a 37 degree celsius water bath until fully thawed. Then, disinfect the tubes using 75%alcohol. Place the disinfected tubes on an ultra-clean table.

Using a pipette, aspirate the cell suspension and transfer it to a 15 milliliter centrifuge tube. Centrifuge the tube in a low speed centrifuge at 250 G and four degrees celsius for five minutes. Meanwhile, prepare four 10 centimeter culture dishes and add nine milliliters of medium to each dish.

Mark the name, cell type, generation, and the experiment date on each dish. Then, preheat them in a 37 degree celsius thermostatic cell incubator. Once the five minute centrifugation is over, disinfect the centrifuge tube before moving it to an ultra-clean table.

Using a pipette, remove the supernatant and add four milliliters of medium to achieve the desired cell concentration. Gently agitate the solution until it is evenly dispersed. Next, add one milliliter of cell suspension to each preheated dish and shake it by agitating the same on a level surface in a cross pattern.

Observe the cells at 40x magnification under an optical microscope before placing the dishes into the 37 degree celsius incubator. On day two, observe the cell state under the optical microscope at 40x magnification before proceeding for exchange medium preparation. After disinfection, transfer the culture dishes to an ultra-clean table with biosafety level two.

Remove the medium from each culture dish. Rinse the dish gently with two milliliters of PBS solution twice and add 10 milliliters of medium to each dish. Transfer the cells to the incubator until they're ready for supernatant collection.

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 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 2, 000 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 ultracentrifugation at 10, 000 G for 30 minutes at four degrees celsius. Then, perform a final centrifugation at 100, 000 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 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.