Hi, everyone. I aim to treat some KC lineages with extracellular vesicles (EVs) from culture media of another treated cells (these ones simulating a pathological state). We have a commercial kit to isolate the EVs (Total Exosome Isolation - Thermofisher), however, I would like to know if someone has experience with this type of experiment and good results using this kit for posterior functional studies. Or in this case, I have to obtain the EVs only by differential centrifugation?
Thank you very much, and futher suggestions will be appreciated! :)
Hi Amanda, are you interested only in exosomes,, or more generally into the population of extracellular vesicles (EVs) that can be released by the cells? You could consider doing a standard centrifugation of the conditioned medium at 2'000 xg for 20 min at RT, and then look at the supernatant by a technique like DLS to have an idea of the mean size of the the EV populations you have. You may very well have EVs population in the 200-300 nm size. By doing further ultracentrifugation (UC) at 20'000 x g or 100'000 xg you could pellet the EVs and test their functionality. All these steps can ve done under aseptic conditions to maintain bacterial sterility which makes things easier for further cell experiments. Please also test the growth mediim with FBS that you use for the cell cultures to make sure it does not contain itself FBS-derived EVs that can interfere with your cell cultures assays. Of course you can always compare the UC-derived EVs to the exosomes isolated by the Total Exosomes Isolation kit. I hope this helps. Thierry
Hi Amanda, are you interested only in exosomes,, or more generally into the population of extracellular vesicles (EVs) that can be released by the cells? You could consider doing a standard centrifugation of the conditioned medium at 2'000 xg for 20 min at RT, and then look at the supernatant by a technique like DLS to have an idea of the mean size of the the EV populations you have. You may very well have EVs population in the 200-300 nm size. By doing further ultracentrifugation (UC) at 20'000 x g or 100'000 xg you could pellet the EVs and test their functionality. All these steps can ve done under aseptic conditions to maintain bacterial sterility which makes things easier for further cell experiments. Please also test the growth mediim with FBS that you use for the cell cultures to make sure it does not contain itself FBS-derived EVs that can interfere with your cell cultures assays. Of course you can always compare the UC-derived EVs to the exosomes isolated by the Total Exosomes Isolation kit. I hope this helps. Thierry
Hello, Amanda.
I recommend Thierry Burnouf’s answer above. May I add that the heterogeneity of EVs is a crucial issue, especially if you are trying to define a protocol for future translational studies. Check, for example the article below, and further studies from Vilma Martins’ group at the A.C. Camargo Cancer Center in Sao Paulo.
Hajj GN, et al. The unconventional secretion of stress-inducible protein 1 by a heterogeneous population of extracellular vesicles. Cell Mol Life Sci. 70: 3211-3227, 2013.
Good luck eith your experiments.
Thank you very much for your helpful responses!
I am interested in exosomes and microvesicles, that are generally studied. Actually, I already use both type of EVs, considering that the exosome isolation kit yields sample enriched (but not only) for exosomes. However, I was worried about a possible interference of PEG in EVs-treated cells.
Hi Amanda.
Please check the article :
Extracellular vesicles as a platform for membrane-associated therapeutic protein delivery. JOURNAL OF EXTRACELLULAR VESICLES, 2018
VOL. 7, 1440131. https://doi.org/10.1080/20013078.2018.1440131
You will find a rich discussion in the section about large-scale production of EV-based therapeutics.
Good luck
Hi guys, I read this question/answers and I was just wondering - how can I be sure that FBS-derived EVs will not effect my results since I am culturing the cells in the presence of 10% FBS or 2% horse serum. Is there any way how to avoid this - any steps to do?
Thank you!
Hi Veronika, it is conceivable that FBS-EVs may have an impact of the cell growth. Whether this will affect your results depends of what you are looking at for your research. If your interest is to recover the cell supernatant (conditioned medium) to isolate your cells-EVs, then you will have a problem to differentiate them from FBS-EVs. In this specific case it would be wise to buy FBS free of EVs (this is commercially available) or to do an ultracentrifugation step of the FBS to pellet the EVs, as described above. Regards, Thierry
we recovered exosomes with Total exosome isolation reagent (Invitrogen) from various sources, and applied them to misc recipient cells, as well as injected mice. No toxic effects were observed. Keep in mind- you want to make sure that you are utilizing physiologically relevant concentrations; dont climb too high as after certain level - any "foreign" material could be inducing toxic, off-target and other effects in vitro/in vivo
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