Abstract
Cells are known to release different types of vesicles such as small extracellular vesicles (sEVs) and large extracellular vesicles (LEVs). sEVs and LEVs play important roles in intercellular communication, pre-metastatic niche formation, and disease progression; both can be detected cell culture media and biological fluids. sEVs and LEVs contain a variety of protein and RNA cargo, and they are believed to impact many biological functions of the recipient cells upon their internalization or binding to cell surface proteins. It has recently been established that standard isolation techniques, such as differential ultracentrifugation, yield a mixed population of EVs. However, density gradient ultracentrifugation has been reported to allow the isolation of sEVs without cellular debris. Here, we describe the most common methods used to isolate sEVs from cell culture medium, mouse and human plasma, and a new technique for isolating sEVs from tissues as well. This article also provides detailed procedures to isolate LEVs.
Highlights
Cells are able to communicate with each other through the shedding of extracellular vesicles (EVs), which include small extracellular vesicles and large heterogeneous vesicles[1,2,3,4]. sEVs are secreted in vivo by various cell types into biological fluids, including blood, urine, and in vitro in cell culture medium[5]
Either collect the supernatant after 24 h or use medium supplemented with sEV-depleted fetal bovine serum (FBS). 4
Most protocols for Size-exclusion chromatography (SEC)-based purification of EVs still involve differential ultracentrifugation or other pre-clearing steps using high performance liquid chromatography in which the sample is pumped through the column and the fractions collected at pre-set intervals based on either a set amount of time or volume
Summary
Cells are able to communicate with each other through the shedding of extracellular vesicles (EVs), which include small extracellular vesicles (sEVs) and large heterogeneous vesicles[1,2,3,4]. sEVs are secreted in vivo by various cell types into biological fluids, including blood, urine, and in vitro in cell culture medium[5]. Cells are able to communicate with each other through the shedding of extracellular vesicles (EVs), which include small extracellular vesicles (sEVs) and large heterogeneous vesicles[1,2,3,4]. SEV development starts in multivesicular bodies, which are formed by the intraluminal inward budding of endosomes. These intracellular vesicles frequently fuse with the plasma membrane of the cell, to be released into the extracellular space. We describe the procedures for successfully isolating sEVs from cell culture medium, plasma or tissues using differential ultracentrifugation and iodixanol density gradients. We illustrate the isolation of LEVs from cell culture medium using differential ultracentrifugation and iodixanol density gradients. We discuss the isolation of sEVs using an immunocapture technique
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