I wish to isolate exosomes from MSC conditioned medium and would like to know what is the appropriate temperature for storing it in case i am not proceeding with exosome isolation immediately after conditioned media collection .
1) Can conditioned media be stored at 4C/-20C for a week prior to exosome isolation. Will it damage the exosomes or degrade the biomolecular cargo present within them.?
2) I am aware of the fact that conditioned media can be stored at -80C , but somehow feel uncomfortable doing the same as i think thawing conditioned media from -80 to 4C might damage exosomes resulting in poor exosome yield .
So i need help in two things
1) what is the best temperature (4C/-20C) for short term storage (1-2 weeks) of conditioned media that would cause minimal exosomal damage ?
2) if storing at -80C , what is the best procedure for thawing conditioned media without damaging exosomes..
Thank you
Hi,
Depends on the usage. If you would like to use exosomes within 24-48h for physiological purposes like injecting mice with exo-you should keep it on ice at 4C. For collecting protein from Exo, it is better to keep at -20C. For RNA/microRNA extraction-it is better to keep it at -80C.
Good luck,
Zack
It is better you extract exosomes immediately after you collect the CM. This avoid EVs degradation. After the extraction, keep it always at -80°C. Thaw in ice at 4°C.
thank you for you suggestions Zakaria Y. Abd Elmageed and Lítia Alves de Carvalho.
I agree with Litia, you might damage the membrane after freeze n thaw that might give you difficult time to isolate, and even you isolate exosomes but creat doubts in your Isolation to get reliable data from your planned experiments.
Anytime you can keeo your sample from degrading I think its best.
-80. U may consider freezing like whole cells and use DMSO. but do confirm this wont affect your downstream applications
I am not sure what you want to do with your prep but I keep exome prep in fixative for up to a week at 4°C before performing electron microscopy.
I think it depends on what you want to do with the exosomes. In our hands we are using exosomes as a delivery platform for membrane bound enzymes and we found that freezing the conditioned media at -20 gave a very similar yield of enzyme activity when compared with isolating exosomes directly (no freeze-thaw).
J
Thanks everybody for your answers. I was more worried about the effect that freezing would have on the membrane integrity and molecular cargo of exosomes present in conditioned media. Jonathan's answer indicates that storing conditioned media at -20 doesn't cause exosomal membrane damag and simultaneously preserves protein structure ,as he is obtaining similar yields of enzymatic activity in exosomes isolated from fresh conditioned media as well as the one stored at -20.
few more questions to jonathan as well as people who are following this question. I have to perform functional assays with the exosomes after i isolate them from conditioned media.
1) Can the conditioned media be stored at - 20 for at least a week.
2) if yes , will it have any substantial effect on exosome yield as compared to exosomes isolated from freshly extracted conditioned media.
3) exosomes also contain miRNA which are potent regulators of gene expression. what about their structural and functional integrity. Will storing conditioned media at -20 damage them. This is important as i have to perform functional assays .
Thank you
Hi,
You can keep exosomes at 4°C 2 days, after 2- 3 days exosome size will be decreased, you should store exosomes at −20 °C, size remains constant at this temperature. However if you want to isolate miRNAs, -80 is suitable temp. to store exosomes, -20 °C also keeps exosomes miRNAs stable but I recommend don't take this risk.
Good Luck
Ozge
Once having done a 100.000 g ultracentrifugation and having re-suspended the pellet containing exosomes, you can store your sample at -80C.
we typically process it fresh, but cell media can be stored at 4C for up to one week- and no major changes with exosomes/EV
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