There is an increasing appreciation for the heterogeneous nature of extracellular vesicles (EVs). In addition, two nonvesicular extracellular nanoparticles (NVEPs), exomeres and supermeres, have been discovered recently that are enriched in many cargo previously ascribed to EVs. The EV field has largely focused on EV isolation and characterization, while studies on NVEPs are limited. At this juncture, it is critically important to have robust and reliable methods to separate distinct populations of EVs and NVEPs to assign cargo to their correct carrier.
Researchers at the Vanderbilt University Medical Center provide a comprehensive step-by-step protocol for sequential isolation of large and small EVs, nonvesicular fractions, exomeres and supermeres from the same starting material. They describe in detail the use of differential ultracentrifugation, filtration, concentration and high-resolution density-gradient fractionation to obtain purified fractions of distinct populations of EVs and NVEPs. This protocol allows assignment and enrichment of a biomolecule of interest to its specific extracellular compartment. Compared to other isolation methods, our protocol has unique advantages, including high purity and reproducibility, with minimal expertise required. The protocol can be applied to purification of EVs and NVEPs from cell culture medium and human plasma and requires ~72 h to complete. Adoption of this protocol will help translational investigators identify potential circulating biomarkers and therapeutic targets for a host of human diseases and allow basic scientists to better understand EV and NVEP biogenesis and function. Overall, this protocol will allow those interested in isolating EVs and extracellular particles to advance scientific inquiry to answer outstanding questions in the field.
Overview of steps for isolation of EVs and NVEPs from cell-conditioned medium
Schematic for isolation of large EV pellets (lEV-Ps), small EV pellets (sEV-Ps), exomeres and supermeres. Serum-free conditioned medium is centrifuged (500g and 2,000g) to remove dead cells, cellular debris and apoptotic bodies. The lEV-P is obtained after centrifugation of the supernatant at 10,000g centrifugation for 40 min. The leftover supernatant is first concentrated and then subjected to ultracentrifugation at 167,000g for 4 h to obtain the sEV-P (washed one time in PBS by ultracentrifugation at 167,000g for 4 h). The supernatant from the previous step is centrifuged at 167,000g for 16 h to isolate the exomeres (washed one time in PBS by ultracentrifugation at 167,000g for 16 h). The supernatant from the previous step is centrifuged at 367,000g for 16 h to isolate supermeres. b, Representative photographs of the most important steps during the concentrator procedure from a.