We are looking for a cost-effective way to grow adherent cells (HCT116, US2OS), in amounts of 1*10^10, for subsequent isolation of nuclei and preparation nuclear extracts per Dignam 1980. Currently, we scrape cells from about one hundred 100 mm dishes, and get very good results. We now need to scale this up 10-20 fold. It appears that hollow fiber modules may work for growing the cells economically. However, I found limited evidence from the literature on how toquantitatively recover adherent the cells from the modules. I have reservations about using trypsin. Can cells be scraped off the fibers? We would be happy with 50% recovery, as long as the recovered cells remain relatively intact (i.e. not much worse that the state they are in after scraping).
We can also modify modules, or make our own, if special surface treatments/special fibers are needed.
You can wash away most of the trypsin by pelleting the cells and resuspending them. The small amount of residual trypsin can be inhibited by adding PMSF or other serine protease inhibitor.
You're probably going to have to use trypsin + mechanical agitation. You might also try using 0.5 mM EDTA in citrate saline + mechanical agitation. I used this method to release adherent CHO cells from glass when I didn't want to use trypsin.
Article ATPase activity of purified and reconstituted P-glycoprotein...
Thank you, Adam. If one does use trypsin, can it get reliably washed away prior to cell lysis (our protocol calls for hypotonic swelling and douncing).
FiberCell systems mentioned Accutase, I was going to try that as well.
You can wash away most of the trypsin by pelleting the cells and resuspending them. The small amount of residual trypsin can be inhibited by adding PMSF or other serine protease inhibitor.
Adam -- many thanks, thank you. We do use PMSF and the complete inhibitor mix (redundant, perhaps) during the preps as a default, so, I will give that a shot.
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