I culture human iPSC in monolayer for my research, kept in 6 well plates coated with either matrigel or laminin, and kept in either mtesr or iPSC brew (sometimes a 1:1 mixture). Previously they have always passaged fine. Recently the very same line doesn’t passage well because when centrifuged, they don’t form a cell pellet. I am lifting them using 3 minutes of relesr in the incubator followed by scraping. They lift fine; this isn’t the issue. But when I centrifuge them at 300g for 5m (what I’ve always used) they remain suspended and do not form a pellet. I even will centrifuge again at up to 800g for 6 min - still no pellet. Any ideas why?! Please assist!
Sometimes cells don't pellet well if there is DNA released from dead cells. What do they look like when they don't pellet? A wispy looking aggregate is often a sign of a DNA problem.
If this is the case you could try a DNAse treatment. You could also try something to reduce cell death like scraping more gently. Or instead of scraping after Relesr treatment, gently wash the cells off the plate with media.
Of course this assumes it is not something basic like the centrifuge note working properly or being set to RPM instead of g (seen this happen quite a few times).
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