I have to isolate exosomes and perform a few functional assays with them. I cannot afford to isolate exosomes every time for performing each single functional assay due to financial constraints. So I want to know how many cycles of freeze thaw can purified exosome preparations withstand without compromising their quality. And which freeze thaw procedure would minimise the damage
1. room temp to -80C
2. room temp to -20C
I want to store and use the exosomes for at least a month or two.
Hi, we produce exosomes on a larger scale and store them in -80C for later use. At least in terms of morphology and the detection of classical exosomal markers (flow cyt, WB, immunolabeling and TEM) we can not see any difference between exosomes exposed to freezing and thawing and exosomes freshly made. However, the functionality of the exosomes might have to be looked into.
If possible, exosomal preparation should be used fresh without freezing. The exosomal proteins tend to aggregate and activate certain responses that could complicate the future interpretations of results.
If you have to freeze them; -80 degrees only, you should only defrost once.
-80 degrees C would contribute to only a small loss in exosomes, however, you may not be able to account for any functional changes, also, lysed extracellular vesicles may be pro inflammatory.
Also, remember to assess their functionality, you need to perofrm any experiments at body temperature.
Thank you all for sharing your respective views ,will definitely keep them in mind.
Ketil : for how long do you store your exosome preparations ?
divaker : i will try using them immediately on isolation as and when possible.
Dan : As advised by you i will store them at -80. I will make aliquots of the exosome preparations in order to avoid repeated freeze thaw cycles .You made a very valid point which i never thought of that lysed EMV's could cause inflammation .
Agree with Ketil , I store them in allocates at -20 , and functioned well in at least one month ... although fresh made exosome is always better choice for functional study .
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