Exosomes are small membrane bound vesicles between 30 and 100 nm in diameter of endocytic origin that are secreted into the extracellular environment by many different cell types. Exosomes play a role in intercellular communication by transferring proteins, lipids, and RNAs to recipient cells. Exosomes from human cells could be used as vectors to provide cells with therapeutic RNAs. Here researchers from the University of Gothenburg describe how exogenous small interfering RNAs may successfully be introduced into various kinds of human exosomes using electroporation and subsequently delivered to recipient cells. Methods used to confirm the presence of siRNA inside exosomes and cells are presented, such as flow cytometry, confocal microscopy, and Northern blot.
Exosome isolation workflow. Transfer cell culture medium (step 1a) or plasma after centrifugation of buffy coat (step 1b) to a 50 ml tube. Pellet the cell debris at 3000 rpm, 15′ at 4 °C and transfer to an ultracentrifuge tube, centrifuge at 16,500 × g, for 30 min at 4 °C and collect the supernatant being careful not to detach the pellet containing cell organelles and cell compartments. Transfer the supernatant in a new ultracentrifuge tube using a 0.2 μm filter and pellet the exosomes at 120,000 × g, 70′ at 4 °C; cut the sealed top of the tube and remove part of the supernatant with a syringe, then cut the tube in half and pour the rest of the supernatant. Let air-dry the exosome pellet and resuspend with PBS; store the resuspended exosome sample at −80 °C or use directly for the application of interest