I would like to know the role of CO2 in terms of attachment of ipsc to Geltrex/Matrigel/Vitronectin matrix. Will the cells detach as clumps or die as single cells if there is improper CO2 levels in the incubator.
What are the other possible reasons for ipsc cells getting detached during culture.
"Proper" CO2 level is only in relation to the resulting pH of the media. If the CO2 level is wrong then the pH is wrong if beyond the buffering capacity of the media. Most biological systems only function within a narrow pH.
I suspect the real question is in your last sentence. If you are not getting proper attachment, or getting subsequent detachment, then the first suspect is your ECM coating.
Ron:- I have observed the colonies look fine on the first day of attachment but tend to die as single cells or as clumps subsequently. Second observation is the cells tend to survive if passaged to same matrix with more than usual cell death but are dying as single cells or clumps by day2 if transitioned to different matrix. The cells used to attach before even when we transitioned to different matrix. We have checked different ECM-VTN, GTX, Matrigel. Facing the same problem for all.
Bhaskar:- Do you have any suggestions on how to check and rectify the CO2 issue if that is the ISSUE as the incubator shows 5%CO2. We also gave the samples for contamination check for which all the results were negative.
Thanks in advance.
Yes, of course CO2 is required for more than pH maintenance. My answer was given in response to the context of the original question. Calibration of incubator temperature and CO2 controls should be a routine part of incubator maintenance.
Have you previously successfully cultured iPS cells or has this been an ongoing problem from the beginning?
Bhaskar: I shall check with the gas analyzer. If the cells were differentiated it would show a lower cytoplasmic:nucleus ratio. My labmate has a set of cells growing on VTN when he tried to change the matrix to Laminin , the cells died and lifted off. I feel they might not be because of differentiation.
Ron: This problem is happening only for past few weeks now. Everything was well standardized and working fine till.
The funny thing is the same cells are being grown in GMP setting which look fine over there, while in the normal lab atmosphere this problem is seen. All the reagents used over there and here are the same.
Hey guyz, Thanks for your suggestions. We found out that the cultures were getting affected as we had changed our plastic ware reagent to a different company due to which all the sensitive cells were dying. Just be careful while using the plasticware items.
Was the new plasticware not tissue culture treated? Given that you were using ECM coatings the plastic had to have been very bad.
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