A Magnetic Separation-Assisted High-Speed Homogenization Method for Large-Scale Production of Endosome-Derived Vesicles

In this study, we try to address a critical unmet need in exosome research, which is developing a large-scale and reproducible production method for exosome mimetics. Due to the low yield of native EVs, exosome mimetics, EMs, has been produced as surrogates by methods such as real exclusion and centrifugation-based filtration. Certainly, most exosome mimetics are from the direct exclusion of whole cells, which demonstrate similar biologic functions, but are inevitably contaminated by cell debris and organelles.

Extracellular vesicles have attracted significant attention in biomedical research, especially in disease treatment. However, traditional EV isolation techniques like ultracentrifugation, inside exclusion, catamental glyphic start from low yield, poor reproducibility of plow down quality, and require expensive and engineer-intensive production equipment, that cannot meet the clinical demand for EVs. Our study provided a simple and no-cost method for scaling up production of exosome mimetics.

We take advantage of unique biological finding that magnetic nanoparticles can be only internalised within cell endosomes, not other organelles. Thus, we can formulate the endosomes into uniform nanovesicles. This method effectively improve EM yield and reduces cost.

To begin, culture the cells in DMEM complete medium containing 10%FBS and 5%penicillin streptomycin in a six well plate. Incubate the culture overnight at 37 degrees Celsius and 5%carbon dioxide. The next day, co-culture the cells with 10 nanomolar polylysine-modified iron oxide nanoparticles, or IONPs.

Incubate at 37 degrees Celsius and 5%carbon dioxide for 12 hours. After incubation, add 0.5 milliliters of trypsin per well for three minutes to digest the cells. Then, add one milliliter of complete medium per well to stop the digestion.

Centrifuge the cell suspension at 1, 000 G for five minutes and discard the supernatant before resuspending the pellet in PBS. Then, resuspend the pellet in eight milliliters of freshly prepared hypotonic solution for 15 minutes. Transfer the cell suspension to a glass test tube and using a glass homogenizer, apply 20 shocks at 1, 000 RPM to release the organelles.

After homogenization, transfer one milliliter of the cell suspension to a 1.5 milliliter microcentrifuge tube. Place the tube in a magnetic separator for one hour to fully separate IONP-loaded endosomes from other organelles and cell debris. To collect endosomes, discard the liquid from the tube and add three milliliters of PBS to resuspend the endosomes.

Then, use a pipette gun to blow to resuspend the brown pellets found on the contact surface of the tube, next to the magnetic frame. For high-speed homogenization, transfer the endosome solution to a 15 milliliter centrifuge tube. Place the icebox under the tube and then place the tube on the high-speed homogenizer.

Place the 10 milliliter probe into the tube without touching the bottom. On the homogenizer screen, adjust the speed to 140 G and set the time for five minutes. Press the okay button followed by the start button.

After high-speed homogenization, transfer the nanovesicle suspension to a 1.5 milliliter tube and place the tube in a magnetic separator for one hour. Finally, collect the liquid containing the required exosome mimics, or EMs, without disturbing the surface next to the magnetic frame. The morphology of BMSC-EMs analyzed by NTA and TEM showed a typical bowl-shaped, vesicle-like structure delimited by a lipid bilayer.

Both BMSC-EMs and 293T-EMs had similar hydrodynamic diameter to native EVs. Western blotting results showed that BMSC-EMs contain the same protein biomarkers as EVs, and had almost no plasma membrane contamination. The yield of EMs was significantly higher than native EVs prepared by ultracentrifugation.

The EMs had a similar protein composition to native EVs.