I am currently struggling to get non-adherent or dead hepatocytes out of the system after seeding them into a microfluidic chip. I have already tried to increase cell viability prior to seeding using percoll separation, but unfortunately this did not bring the desired success.
The majority of the cells adhere very well to the pre-coated membrane and look morphologically normal, however, there is always increased cell debris on top of the healthy hepatocytes, which can no longer be flushed out of the system, as they are very adherent. The hepatocytes get a gravity wash after 4h of seeding and are overlaid with matrigel 24h afterward. We already tried mild trypsinization and faster flushing, but they just won't come out.
What can I do to increase the viability of the hepatocytes and to remove the dead cells?
I would be very grateful for any suggestions and ideas!
Hi Tim,
Have you tried using dielectrophoresis or acoustophoresis to separate the live cells from dead cells/debris? These methods can be cumbersome, but if you're dealing with a small volume of cells they could be OK.
Also check out this paper, they've developed a method for removing non-viable cells and debris from viable cells using inertial microfluidics: https://pubs.rsc.org/en/content/articlelanding/2018/lc/c8lc00250a
Also, it might be worth trying to encapsulate the cells in the ECM material prior to loading your device. If you have a hard time reliably getting Matrigel, it is worth checking out OkaSciences. They make Matrigel equivalents and ship next day! https://okasciences.com/shop/okamatrix/
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