This protocol provides a rigorous and reproducible technique for extraction and isolation of small extracellular vesicles from whole tissue specimens for downstream ex-vivo analyses. With amongst the highest levels of purity currently achievable, this method facilitates the direct morphologic, immunophenotypic, and deep characterization of interstitial vesicles secreted into various tissue specimens, including tumors. Here, we demonstrate the isolation of tissue EVs from brain and lung tumor specimens.
However, this technique can be applied to studies using diverse, benign, or other tumor specimens as well. To begin, prepare 10 milliliters of dissociation buffer in Hibernate-E medium for every 0.4 to 1.0 gram of tissue. Add whole fresh or frozen tissue to the buffer in a 50 milliliter tube, and incubate in a warm water bath at 37 degrees celsius for 20 minutes.
After this, add protease and phosphatase inhibitors for a final 1x concentration in the dissociation buffer. Pour the solution with the tissue into a loose fit downs homogenizer. Use approximately 30 slow strokes per sample to gently dissociate the tissue.
Then, transfer the dissociated tissue and buffer solution to a 50 milliliter conical tube. Centrifuge at 500x g and at four degrees celsius for five minutes to pellet the cells and the remaining fibers or cohesive tissue fragments. Transfer the supernatant to a clean 50 milliliter conical tube, and centrifuge at 2, 000x g and at four degrees celsius for 10 minutes to pellet and discard the large cellular debris.
Transfer this supernatant to a clean 50 milliliter conical tube, and centrifuge at 10, 000x g and at four degrees celsius for 40 minutes to pellet any undesired larger vesicles or small apoptotic bodies. Decant the supernatant through a 0.45 micrometer filter into a clean 12 milliliter ultracentrifugation tube. Next, ultracentrifuge the sample at 100, 000x g and at four degrees celsius for two hours, to pellet small EVs.
Decant the supernatant and leave the ultracentrifugation tubes inverted for five to 10 minutes, tapping frequently to remove any residual liquid on the sides of the tubes. Then, resuspend the EV pellet in 1.5 milliliters of 0.25 molar sucrose buffer. Cover the tubes with Parafilm, and then vortex the EVs into solution.
Rock the ultracentrifuge tubes for 10 to 15 minutes at room temperature. And then vortex once more. Briefly centrifuge the tubes at a speed less than 1, 000x g to recover the liquid suspension at the bottom of the tube.
If needed, store the suspension at four degrees celsius overnight. First, add 1.5 milliliters of 60%iodixanol to the 1.5 milliliters of the sucrose tris buffer that contains the EVs to create a final solution containing 30%iodixanol. Pipette up and down several times to mix the solution thoroughly.
Transfer this solution to the bottom of a 5.5 milliliter ultracentrifugation tube. Next, mix the 60%iodixanol stock with ultra pure water to prepare at least 1.5 milliliters of both a 20%and a 10%iodixanol solution. Using a syringe and an 18 gauge needle, measure 1.3 milliliters of the 20%iodixanol solution and carefully layer it on top of the bottom gradient.
Keep the bevel of the needle in contact with the inside of the tube, just above the meniscus and add the solution drop wise to avoid mixing the layers at the density interface. Then, layer 1.2 milliliters of the 10%iodixanol solution on top of the 20%layer, using the same technique. Carefully balance and load the ultracentrifugation tubes into rotor buckets.
Set the acceleration and deceleration speeds of a swing bucket rotor to the minimum rates, and centrifuge at 268, 000x g and at four degrees celsius for 50 minutes. While the sample is being centrifuged, label 10 1.5 milliliter microcentrifuge tubes for each sample that will correspond with fractions one through 10 of the density gradient. Once the centrifugation is complete, gently remove the tubes from the rotor buckets and place them into a stable holder.
Pipette 10 serial fractions of 490 microliters from the top of the gradient into the corresponding tubes. Using a refractometer, measure the refractive indices of the fractions. Then, transfer each fraction to a clean 12 milliliter ultracentrifugation tube.
Add five milliliters of 1x PBS to each tube and pipette up and down slowly to mix. Add an additional six milliliters of 1x PBS to the top of the tube, and carefully mix again. Ultracentrifuge the tubes at 100, 000x g and at four degrees celsius to re-pellet the small vesicles.
Decant the supernatant and tap the tubes dry before lysing the vesicles for protein analysis or reresuspending the EVs for morphologic analysis. To lyse the EVs for protein analysis, add 40 microliters of strong lysis buffer containing protease inhibitor to the EV pellets. Place Parafilm over each tube, and vortex vigorously.
Next, rock the tubes for 20 minutes at room temperature and vortex again. Briefly centrifuge the sample at 1, 000x g for 30 to 60 seconds to recover the entire sample volume. Transfer each sample to a new 1.5 milliliter microcentrifuge tube, and store it at a temperature between 20 and 80 degrees celsius until ready for further processing.
To prepare the purified lysates for immunoblot analysis, add 5x Laemmli sample buffer to the samples for a final concentration of 1x. Boil the sample at 95 degrees celsius for five to 10 minutes. Then, load an equal volume of fractions one through 10 into a 10%SDS page gel.
Load an equal mass of tissue homogenate. Perform electrophoresis and Western blot analysis to confirm EV proteins in the purified lysates and compare relative EV abundance in fractions. In this study, extracellular vesicles are extracted and purified from whole tissue.
Following ultracentrifugation of the 10 to 30%iodixanol gradient, a population of light EVs can be seen migrating up to fraction two, while a population of dense EVs can be seen migrating up to fraction five, depending on tissue type. Representative immunoblots of the gradient fractions exhibit the efficient separation and purification of small tumor derived EVs in fraction five. Notably, lung tumor specimens appear enriched in dense EVs compared to light EVs that were previously harvested from whole brain tissue.
Representative nano particle tracking analysis and electron microscopy of tissue-derived vesicles in the predominant vesicle containing fraction demonstrate the enrichment and preservation of whole vesicles. Consistent with the known size and structure of small EVs. Interstitial EVs represent promising targets for the development of novel diagnostic or prognostic biomarker assays.
This technique provides researchers with tools for the extraction and purification of vesicles for downstream utility. Following extraction of whole or lysed tissue EVs, broad characterization of vesicles including proteomic, genomic, and lipidomic analyses may be performed. Additionally, samples may be used for more targeted approaches.
Vesicles isolated directly from tissue specimens can provide further insight into disease mechanisms, including tumor genesis, and may provide important diagnostic or prognostic tools in the future.
