Extracellular Vesicle Extraction: A Technique to Isolate Extracellular Vesicles from Whole Tissue Specimens Using Differential Centrifugation
Transcription
In whole tissues, extracellular vesicles or EVs are released by the cells into the interstitial fluid. These include apoptotic bodies, microvesicles, and exosomes. The EVs play an important role in maintaining cell-to-cell communication.
To extract EVs, begin by taking a whole tissue sample.
Treat the tissue with an appropriate dissociation buffer. The components of dissociation buffer disintegrate the extracellular matrix present in the tissue, eventually loosening the cells.
Transfer the suspension to a homogenizer and mechanically disrupt the tissue further to dissociate it and release the cells.
Next, centrifuge the homogenate at low-speed to pelletize the cells and tissue fragments while the EVs remain in the supernatant.
Take the supernatant into a fresh tube. Centrifuge at high speed to pelletize most of the larger-sized apoptotic bodies, leaving the smaller-sized EVs in the supernatant.
Collect the EV-containing supernatant into a syringe. Filter the components to remove any remaining apoptotic bodies and collect the small-sized EV population.
Centrifuge the filtrate to obtain the EVs in the pellet. Discard the remaining buffer and resuspend the EV pellet in a sucrose solution. This solution creates an isotonic environment for the EVs to retain their morphology.
Now, store the EV suspension at four degrees Celsius until further use.
