Hi ,
I have been working with iPSC cell lines and hESCs for 6 years with Matrigel and mTESR1.
I have recently started working with iPSC cells with vitronectin matrix and Stemflex media . However I have noticed a lot of dead cells along with black dot like bodies . It doesn't look like any kind of contamination and we have tested the cells for mycoplasma and the cells seem healthy with very less differentiation. I have noticed that the cell debris / death is more when the cells are more confluent. The media is changed on Monday , Wednesday and Friday . Does anyone have experience with this system? I have never noticed anything like this before.
Is this amount of cell death and debris normal?
On a side note :
We usually passage the cells on a Thursday but for some of our cell lines , the passaging starting falling on a Monday ? Does passaging the cell lines after the weekend free feeding affect the cell lines ?
We have also noticed the CO2 and temperature drop to a degree less within seconds of opening the incubator. Could that also be an issue?
Thanks
Hi there,
From the picture you provided, it seems like your cells look healthy but they are detaching very fast due to being overconfluent in short time. I was going through this phase with my hiPSCs when I was using Gibco E8 flex media. With iPSCs and its overall health, you should always change media everyday, I wouldn't trust this Flex media gimmicks.
Switch to either E8 media or mTesr1 media (no Flex and change the media everyday), they work best with hiPSCs and also try to use Matrigel if possible. Try to maintain the passaging in 3 to 4 days, even if they are not confluent until it follows that routine. This works best for my differentiation pattern. Also to increase the post passage/thaw viability, use ROCK inhibitor for the first 24 hrs of the passaging and thawing. After 24 hrs. change the media without Rock inhibitor, this increases post passage survival rate.
I hope this will make a difference!
Good luck
We had a similar issue for our neural stem cells. We changed some media components and the black debris went away. I think it is a sign that the cells are not "happy"
Hi,
I have the same issue. Could it be a contaminant? Cells are in Brew XF, Matrigel coating.
Thanks a lot.
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