Hello,

You need to culture large number of cells for collection of exosome fractions from media. I did it by growing cells in 100 cm dishes (20 such dishes, each with 10 ml media were used) by seeding 1.5 million cells initially and allowing them to attain 60 percent confluency. Thereafter media was changed with fresh one and cells allowed to grow for 48 hours. After 48 hours, conditioned medium was collected and centrifuged at 500 rpm to pellet down the cell debri. Finally the supernatant from previous step was subjected to ultra centrifugation at 1,20,000 rpm for an hour to pellet down the exosomes. The purity of exosomes may be checked by subjecting the pellet for protein extraction and doing a western blot with exosome markers (i did it with CD 63). 

Remember to use completely vesicle-free media, preferably serum-free, as you will otherwise isolate bovine RNA that for the most part cannot be distinguished from RNA of your species of interest. If your goal is to isolate exosomes, you will not obtain reasonably pure exosomes by doing only a slow first spin to remove cells. At a minimum, sequential centrifugations at higher g are needed to remove cell debris, apoptotic bodies, and larger vesicles. Showing that a putative exosomal marker is present is also not the same thing as demonstrating that co-purifying complexes are absent!

Article Current Protocols in Cell Biology

Article Minimal experimental requirements for definition of extracel...

You might be interested in this product from Qiagen. They claim to isolate both miRNA and RNA from exosomes and other extracellular vesicles:  http://www.qiagen.com/us/products/catalog/sample-technologies/dna-sample-technologies/genomic-dna/exorneasy-serum-plasma-kits?WT.dcsvid=breanna.symmes@ucdenver.edu&WT.mc_id=QVenRNA1501exoRNeasylaunchEM_NA&cmpid=QVenRNA1501exoRNeasylaunchEM_NA

Hi,

We've grown our cells (MCF7, MCF10A and MDB-MB-231) to 80–90% confluency, washed four times with serum-free media, and then incubated with a minimal volume of serum-free media required to cover the cells.

After four hours of incubation the ‘conditioned’ cell culture media was collected, followed by removal of cellular material by a two-step centrifugation process (1,000×g and 17,000×g) and/or by filtering with 0.22 µm filters to remove large protein aggregates and other cellular debris. We then precipitated EVs from the collected conditioned cell culture media using the ME Kit (Vn96 peptide) by New England Peptide (Exosome Isolation protocol takes less than 45min, No ultracentrifugation needed).

Subsequent miRNA cargo was analysed through NGS. 

**The cell culture media added was prepared with exosome free (Exo-Free) Fetal Bovine Serum (FBS). The Exo-free FBS was prepared by centrifugation of FBS at 100,000×g for two hours at 4°C followed by aspiration of the supernatant without disturbing the exosome pellet.

http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0110443