This protocol is significant because it concentrates extracellular vesicles, EVs, through a combination of technologies, while allowing for separation of virions away from EVs. This method maximizes EV recovery above the current gold standard of ultracentrifugation for multiple downstream analysis and characterization of virus-free EV preps. This protocol is ideal for the study of EV's and viral infections.
This method can be adapted to other virus systems, such as HTLV, Ebola, Zika, and more. I would expect a first time user of this method to struggle with nanoparticle preparation, so we recommend carefully following our specifications. Some optimization may be necessary, depending on the system.
Visual demonstration of this method is important for illustrating the overall work flow and nuances of the protocol, which will reduce time and mistakes for first time users. Start by preparing culture supernatant from infected or transfected cells. Culture approximately 10 milliliters of late log cells for five days at 37 degrees Celsius and 5%carbon dioxide, making sure that all medium reagents are free of extracellular vesicles.
When the culture is ready, pellet the cells by centrifugation at 3, 000 times G for five minutes and discard the pellet. Filter the supernatant with a sterile 0.22 micrometer filter and collect the filtrate in a clean tube. Add an equal volume of PEG precipitation reagents to the filtrate, and invert the tube several times to mix.
Incubate the mixture at four degrees Celsius overnight. After the incubation, centrifuge the mixture at 1500 times G for 30 minutes, to yield a heterogeneous extracellular vesicle, or EV, pellet. Discard the supernatant, and re-suspend the pellet in 150 to 300 microliters of 1X PBS, without calcium and magnesium.
Keep the pellet on ice, while preparing a density gradient. Prepare the iodixanol density gradient medium with 11 density fractions, ranging from six to 18%iodixanol, as described in the manuscript. Mix each tube by vortexing and layer the density fractions into a clean and dry swinging bucket ultracentrifuge tube.
Add the re-suspended EV pellet to the top of the layered gradient and ultracentrifuge the tube at 10, 000 times G, and four degrees Celsius for 90 minutes. Carefully transfer each fraction from the ultracentrifuge tube to a new microcentrifuge tube. Prepare a 30%slurry of nanoparticles for EV fraction enrichment, by mixing equal volumes of NT80, NT82 and 1X-PBS.
Vortex the nanoparticle mixture to ensure homogeneity. Add 30 microliters of the slurry to each density fraction and mix by either pipetting or inverting the tubes. The most critical step is the addition of the nano-articles to concentrate EV's, following the density gradient separation.
Without this, EV recovery is poor. Rotate the nanoparticle containing density fractions overnight, at four degrees Celsius. Then centrifuge the density fractions at 20, 000 times G for five minutes at room temperature.
Discard the liquid and wash the EV pellet twice with 1X-PBS. To prepare the pellet for RNA isolation, re-suspend it 50 microliters of autoclaved, deionized water, filtered and treated with depsy, according to the manuscript directions. The RNA can then be isolated, using a commercial kit.
For analysis with gel electrophoresis, re-suspend the pellet in 15 microliters of Laemmli buffer. Heat the sample to 95 degrees Celsius for three minutes. Repeat the heating step two more times, vortexing gently and spinning the sample down between heat cycles.
Centrifuge the sample for 15 seconds at 20, 000 times G, and load the supernatant directly onto the gel. For best results, limit the amount of particles loaded onto the gel, and run it at 100 volts, to prevent any loaded nanoparticles from entering the gel. PEG precipitation allows for significantly more efficient EV recovery, than traditional ultracentrifugation.
When using 10 milliliters of culture, this approach results in an approximately 500 fold higher yield than ultracentrifugation. This approach also results in an increased isolation efficiency of exosomes, which is evident through higher levels of exosome marker proteins. Western PLA analysis shows, a 3000 fold increase in CD81, a four fold increase in CD63 and a 40 fold increase in CD9.
Furthermore, this protocol allows for downstream studies of EV mediated mechanisms in HIV1 infection, by isolating EV's from verions. Western PLA analysis shows that EV's are found in three fraction populations. While the virus is present in only two of them.
EV's infraction 10.8 through 12 are free of virus contamination. The most important thing to remember is that the nanoparticles are crucial for optimal EV enrichment. Their addition is a must.
Many downstream assays, such as western blot, PCR, pure diomech analysis by mass spectrometry in plate-based cellular assays can be performed. These allow for characterization, mechanistic, and/or functional studies. This technique allows for specific studies that examine EV cargo and functionality without contaminating viral background.
This provides a foundation for studying EV's in pathogenesis and development of potential therapeutics. To overcome the limitations of this technique, and to apply EV preparation and isolation to large volumes, we recommend the use of advanced systems, such as tangential flow filtration. The most hazardous instrument to use is the ultracentrifuge, during the density gradient separation.
Make sure that all centrifuge tubes weight the same, to avoid imbalance and potential rotor failure.
