Hello,

I'm struggling with a problem isolating tumor-derived exosomes. In short:

We are isolating exosomes from concentrated media of HCC cell lines -

cultivation for 72h in DMEM containing 10% exo-free FCS (UC for 18h at 100 000g) - subsequent centrifugation of supernatants at 400g, 4°C for 10min to remove debris -

- filtration using syringe filters (0.22µm) - purification in centrifugal filter devices [Centricons (100 kDa)], multiple runs to reduce the total volume of collected supernatants for ultracentrifugation - subsequent UC at 100 000g, 4°C for 1:30h.

This is the first time we tried an approach with FCS-containing DMEM - previously we cultivated our cell lines in DMEM 0% FCS for 24 hours - unfortunately, we had a major issue, which I think might be albumin-related. Already during purification in the Centricons (100 kDa) we received a viscous isolate in our filter devices (upper chamber), which presumably led to a poorly soluble pellet during ultracentrifugation. Also upon repeated up and down pipetting and letting the pellet sit for several hours at 37°C it could only be partially dissolved.

Albumin with a molecular weight of 66kDa - should actually pass through our filter devices, at least that's what we thought. The question is whether switching our protocol to ultracentrifugation only would solve the problem, as using the Centricons appears to result in increased accumulation of albumin. Additionally, can you think of other ways to get rid of the excess albumin? Maybe using a sucrose cushion

I would be happy for any suggestions