In this paper you have a good summary and protocols of precipitation of exosomes following different protocols:

http://www.ncbi.nlm.nih.gov/pubmed/?term=the+impact+of+disparate+isolation+methods+for+extracellular+vesicles

As for the yield, culturing your cells in a T75 plate you will have aroung 5-6M at confluence and, if you follow the protocol of ultracentrifugation right, you will get aroung 50ug of exosomal protein per flask.

I haven't looked at the paper that Jennifer Perez-Boza (above) sited, but I can recommend that if you are culturing cells with FBS in the media, find a exosome depleated source of FBS.  (There are bovine exosomes in most FBS, and they can confound analysis of exosomes isolated from cultured cells.)

We used a PEG based method.

Best Bernd

Article MSC-derived exosomes: A novel tool to treat therapy-refracto...

Thank you all. you  have been very helpful

So I can't just switch From DMEM supplemented with FBS to serum free DMEM to prepare CM. I need to buy exosome-depleted foetal bovine serum (EDS) right? what if I am using human serum?

Hello Nermeen,

the only thing you have to do with your serum, either normal FBS or any other you use is dilluted 1:2 (in order to decrease serum's viscosity) in your normal medium and centrifugate it overnight at 110.000g, with this, you recover the supernatant and you have exosome free medium without any more problem.

Hope it helped you

Thank you very much Jennifer. That has been very helpful.

I am sorry I have one more question. Do I have to add protease inhibitor to the collected Condition medium?

We never did that. Please also be aware that EV deplition might have a severe effect on the cells and may alter their biological functions. We raise MSCs in human platelet lysat conditioned media. If we reduce the vesicle content of the fresh media by UC, the cells do not grow well anymore. According to our experience and to other reports even over night centrifugation will not deplete EVs completely, in case you like to deplete, you have to check it by NTA. Regarding media EVs, we process the same amount of fresh and conditioned media and compare biological effeects of both fractions. For molecular analyses I would like to recommend density gradient purification. Note EVs harvessted from succhrose are not physiological intact anymore and should not be used in biological assays.

use the Total exosome isolation reagent (from cell media) - it enables fast isolation of highly clean exosomes, comparable to ultracentrifugation protocols. in higher yields. if you need exosome depleted FBS (to grow cells in a "clean"/exo free environment)- Gibco launched this product few months back

MSC exosomes can be collected from approximately 3 early passages (passages 2–3). Once MSC cultures reached 70 % confluence, cells can be cultured for 24– 48 hours in α-MEM containing exosome-depleted FBS. Exosome-depleted FBS should be obtained by over- night centrifugation at 70,000 × g at 4 °C. briefly, centrifuge MSC conditioned medium twice at 500× g for 10 minutes, twice at 2000 × g for 15 minutes and twice at 10,000 × g for 30 minutes. Then transfer the supernatant Ultra-Clear tubes and centrifuged at 70,000 × g for 1 hour at 4 °C in a SW32Ti rotor (Beckman Coulter Inc., Woerden, The Netherlands). Wash the exosome-containing pellet with PBS and centrifug at 70,000 × g for 1 hour. Re-suspend the pellet carefully in 200 μl PBS and use it immediately or store it at −80 °C.

check this out 

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3975389/

Good evening,

I have almost the same problem. I cannot deifine the best density of human bone marrow MSCs in order to isolate a satisfactory quantity of exosomes. I follow the classic protocol with ultracentrifugation. Regarding my western assays my results are not constant all the time. I use as reference proteins alix and cd9 but in some cases they express both of them, in some cases only one of them and in others none. I believe that depends on type/number of cells and on diversity of samples. I started with 500000c/175T (in a total of 4-6 flasks) and then with almost 1*10^6 cells/ 175T (in a total of 4-6 flasks). When cells reach almost 90% of confluncy I change the medium in serum free for 2-3days, I use to trypsinize 2 of harvested flasks to have a measure of the total cells but no change shown in different started cell densities (range about 8*10^6 cells from 2 flasks).

you would suggest me a solution?

Thanks a lot

Aristea

dear Aristea , find the attachment. hope this might be helpful to standardize seeding density for MSC for maximum exosome yield.