Hi all,
I have tried re-plating my HiPSC derived cardiomyocytes into another plate for functional analysis, but when I am passaging the cells form a whole clumpy layer that does not breakdown and keeps on floating in the media. I have tried using cell strainer but the floating/clumpy layer doesn't breakdown. I am not sure if its Matrigel or the cell layer. I use TrypLE as my dissociation reagent.
Can anyone recommend any other dissociation agent that will help this problem?
Dear Darshini Desai
In my experience, Matrigel does not cause such issue. In my hands, the 100micron cell strainer works the best to get single cells .Do you use a similar pore size of the cell strainer?
Also, there could be a few other steps that can affect the efficiency of this process.
Exactly how long do you incubate in TrypLE and in how much volume do you collect your cells when you passage them? Have you tried pipetting them "gently" to dissociate the clumps?
(Too long incubation and/or too harsh pipetting may cause cell death. The DNA released from dying cells can cause more sticky clumps that are harder to dissociate.) In case if this is happening you can try to add DNase in your passaging or change to another dissociation reagent.
Good luck !
Hi Shambhabi Chatterjee,
I use 1ml of TrypLE for 1well of 6 well plate and incubate it for 7 to 8mins at 37 degree. I tired using 70micron strainer, but my issue is the cells comes of in a one single layer and its like a stretchy muscle almost jelly like layer which do not dissociate and pass through any strainer. I collect the cells in 5 ml of media.
I would recommend to try out 5min (not more) incubation with 1ml TrypLE.
Collect the cells by pipetting up and down simultaneously from the well.
Stop the activity of TrypLE by adding equal volumes of medium containing FBS (heat inactivated)
then spin down the cells 200g, 5min at RT
remove the supernatant and resuspend cell pellet in fresh medium (1ml)- mix well by *gentle* pipetting. Then add required amount of medium (4 ml) additionally
this should help you to avoid the clumps
if you still find clumps then strain it through a 100micron filter
Yes, I will try to incubate it more and see if that helps. Thank you so much!!
pls incubate for *shorter* time. not more than 5 min.
(make sure that when you add the TrypLE it is already at RT)
it is difficult to be certain but based on your description I am guessing that the cells are dying , which is causing the cells to stick to freely floating DNA causing the thread like jelly appearance .
So a shorter incubation in TrypLE will most likely solve ur issue.
if needed you may try to add DNase also into your passaging step
good luck !
also you may find some helpful points from this previous discussion on ResearchGate- https://www.researchgate.net/post/How_can_I_reduce_clumping_during_passage_of_iPSC-CM
Yes i will try shorter amount of time to dissociate, also i will try to do metabolic selection if that helps to get more pure colony.
Thanks a lot for all the suggestions!
Hi Darshini,
I've also experienced this issue (using accutase instead of trypLE), including the inability to dissociate the clumps using a cell strainer. My cardiomyocytes had been in culture for 3 weeks before passaging. I suspect that this might play a role too. Have you been able to resolve the issue? I'd like to hear!
Jelmer Faber Hi, I am currently doing the functional assay without using the dissociated cells but eventually i will start on it again for other assay. I will keep you updated!
Darshini Desai Thank you for the response. If, in the meantime, I find a solution, I will let you know! Looking forward to hear from you.
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