I subjected exosome fractions 0.5 mL collected from sephadex CL-2B based size exclusion chromatography to 48 hours freeze-drying. Any suggestions would be highly appreciated.
do you want to check their biological function? or their shape by electron microscopy?
As I believe freeze drying will not cause any harm to exosomes, but if it is related with protein necessary measures should be taken.
freeze - thaw cycles have the risk of rupture the vesicles or create aggregates. most downstream analysis won't suffer from this. but it's something to be aware of in size determination (DLS, NTA), bioassays and EM.
I am not aware about how freeze-drying affects exosomes, but if you want just to preserve exosomes, I would suggest -80ºC. I had exosome (and microvesicle) concentrates for more than two months at -80ºC and I measured them every week. Results showed that neither concentration and neither size distribution were affected by one freezing-thawing cycle (but further freezing-thawing cycles might really affect the sample).
Just to add one more part to my question, my intention is to rupture the exosomes by freeze drying, and not preserve, due to the very specific downstream experiments planned.
Sonication also should help rupturing them. To ensure full rupture you could do a couple freeze-thaw cycles with a short bath sonication in between maybe?
Exosomes can be frozan and thawn without any problems - the exosomal membrane is different than the cell membrane -it emanates from the endosomal membrane and is rich in phospholipids - sphingolipid and ceramid and besides that it is rich in tetraspanin proteins such as CD63, CD81 and CD9 that enforce the stability of the membrane. This makes the exosomal membrane "tough" and resistent to freeze-thaw procedures. Thus, proteins and nucleic acids carried inside the exosomes are well protected by the exosomal membrane. Moreover, the function of the exosomes is not affected by freesing -thawing itself but can be affected by matrix metallo protease (MMP) activity resulting in cleavage of surface proteins - see below.
Importantly, exosomes carry proteins on their surface which can be involved in various biologic mechanisms - i.e. they can carry/express NKG2D ligands that can block the activating NKG2D receptor and suppress cytotoxicity; adhesion molecules that participate in exosome uptake; FasL and TRAIL molecules that are functional and can induce apoptosis etc. etc. It is, therefore, important to protect these molecules from the effect of MMPs and it is recommended (if you are studying molecules expressed/attached to the exosomal membrane) to use MMP inhibitors when freezing exosomes for later use.
Hi Vineesh,
What did you find out when you freeze-dried your exosomes. Did they rupture or not?
Thanks,
Keith
Hi Keith,
Upon freeze drying, we have found the exosomes to form aggregates, but the rupture is not at all efficient, though it happens at a relatively small scale, as seen in nanoparticle tracking analysis. We have since then been using repeated cycles of freeze thaw to rupture the exosomes.
Cheers
Vineesh
Hi Vineesh,
Could you please talk about the repeated freezing-thawing cycles to rupture the exosomes in details, e.g. how many times?
Best,
Junping
related to this question , I would like to know if we want to concenterate the cell culture media (before exsosome purification with sepharose chromatography) is it reasonable to use freeze-drying ?
The cryopreservation including freezing of the EVs, thawing and possible re-freezing after partial usag could be harmful to the EVs. To overcome the dangers associated with freezing, cryopreservation is commonly associated with the addition of one or more “cryoprotectants”... This note is from:
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7006664/
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