An Enrichment Method for Small Extracellular Vesicles Derived from Liver Cancer Tissue

This method allows for the isolation of liver cancer tissue-derived small extracellular vesicles with a purity higher than that achieved by classic, differential ultracentrifugation. This technology is simple, convenient, and easy to operate, which can provide methodologic support for the study of small extracellular vesicles. To begin, remove the tissue sample from the 80 degrees Celsius freezer.

Place approximately 400 milligrams of the tissue into a 10-centimeter cell culture dish on the ice box, and finely mince the tissue with a scalpel. Transfer the tissue to a six-well plate with 1.5 milliliters of the digestive solution. Then, place the plate on the transference decoloring shaker, and incubate at 37 degrees Celsius for 20 minutes for complete dissociation of the liver cancer tissue and release of the small extracellular vesicles, or sEVs.

After incubation, place the six-well plate on the ice box. Add 80 microliters of phosphatase inhibitor and 200 microliters of complete protease inhibitor solution to stop the digestion. Then, transfer the digestive solution to a 70-micrometer cell strainer placed on a two-milliliter centrifuge tube, and filter it slowly to remove any large tissue debris.

Centrifuge the filtrate at 500 g for 10 minutes to remove the small tissue debris, and collect the supernatant in a new two-milliliter centrifuge tube. Centrifuge the supernatant at 3, 000 g for 20 minutes to remove the cell debris. Carefully transfer the supernatant to a new centrifuge tube until the tip of the pipette is about to touch the white debris at the bottom.

After repeating centrifugation of the remaining liquid, pool the supernatant into the tube without disturbing the debris. Centrifuge the collected supernatant at 12, 000 g for 20 minutes, and transfer 900 microliters of the supernatant to a 4.7-millimeter ultracentrifuge tube. Repeat centrifugation of the remaining liquid, and then collect and mix both supernatants into the same ultracentrifuge tube.

Fill the tube completely with PBS. Centrifuge the supernatant at 100, 000 g for 60 minutes. After discarding the supernatant, resuspend the pellet by filling the ultracentrifuge tube with PBS.

After repeating the centrifugation at 100, 000 g for 60 minutes, resuspend the pellet with 50 microliters of PBS. Aspirate the sEV suspension using a one-milliliter sterile syringe, and filter it through a 0.22-micrometer membrane filter into a 600-microliter centrifuge tube. Store the filtrate at 80 degrees Celsius for further analysis.

Use a nanoparticle flow cytometer to determine the size distribution and purity of the sEVs. Check the volume of the cleaning solution. Then, note the difference between the levels of the sheath and the waste liquid.

After 20 seconds of turning on the main power supply of the instrument, turn on the computer. Wait for the beeps indicating that the instrument is properly connected to run the software. Click on Start Up to turn on the camera, laser, and air pump.

Next, click on Sheath Flow, and select Start Up.Place ultrapure water on the loading platform. After four minutes, click on Sample and Boosting, and then select Unload in Sample. After 30 seconds, place the blank tube on the loading platform.

Click on Sample, select Boosting, and then select Unload in Sample. Then, place the tube containing the ultrapure water on the loading platform. Simultaneously, click on Sample and Boosting, followed by Sheath Flow and Purge.

Click on Manual Operation, and place the particle concentration tube on the loading platform. Then, click on Sample, select Boosting for one minute, and simultaneously select 250 nanometer Standard Fluorescent Silica Nanospheres quality control in Sample Information. Select Sampling from Sample, and turn the detector on by clicking on SPCM.

Then, click on Auto Sampling, and enter 1.0 into Sampling Set to fix the pressure at one kilopascal. Click on the toolbar, and select Large Signal. Adjust the horizontal position of the laser, and set the laser to two micrometers.

Then, continuously click on L and R to ensure the signal is strong and uniform. Click on Time to Record to collect the data, which automatically jumps to Buffer when finished. Then, save the data in Nfa File, and click on Sample to select Unload.

Place the cleaning solution tube on the loading platform. Click on Sample, select Boosting, and after one minute click on Sample and Unload. Use ultrapure water to remove the residual cleaning solution from the capillary tip.

Place the particle size standard tube on the loading platform, and click on Sample Boosting for one minute. Select 68 to 155 S16M-Exo quality control in Sample Information, and click on Sample, then Sampling. Next, click on the toolbar, and select Small Signal.

Click on Time to Record to collect the data, which automatically jumps to Buffer when finished. Then, save the data in an Nfa file, and click on Sample to select Unload. After removing the residues with the cleaning solution, place the PBS tube on the loading platform, and perform the steps demonstrated for the particle size standard tube.

Next, clean the capillary tip as demonstrated earlier, and load the sEV sample to analyze the data described earlier for the particle size standard tube. The purified sEVs were examined under a transmission electron microscope, which revealed the presence of sEVs with a cup-shaped morphology. Flow cytometry for the sEVs was carried out.

And the particle size ranged from 40 to 200 nanometers, and the average particle size was 89 nanometers. The purity of the enriched sEVs was found to be 68.32%The protein markers of sEVs such as CD63, CD9, and TSG101 were detected by western blot. GM130 was used as the negative control marker of sEVs, which was found in tissue cells but not in the sEVs.

It is crucial to perform the steps of enzymatic digestion, differential ultracentrifugation, and membrane filtration with utmost care to ensure the purity of small extracellular vesicles.