I have noticed that following retrieval from cryopreservation, the hESCs that we are using experience a cell "crisi" following 3 passages. Although I have heard anecdotal evidence of this being a common phenomena I was wondering if anybody knew if there had been any research carried out as to why?
If not micoplasma, usual issues for cell detachment can be:
Bad matrix - either the plate isn't coated well enough (time, concentration, buffer) or the matrix (I'm thinking things like Geltrx here) isn't a good batch.
Differentiated Cells - if cells differentiate they don't always survive in your maintenance media
Confluence - If the cells get too confluent mine seem to float off in patches
Also, I don't think this would be an issue but are you adding ROCK inhibitor/revitacell etc.? How are you passaging? How have the cells been stored? -80 for a long time can affect them.
Hi,
The hESC cells detach from plate after 3-5 passage when they changed phenotype. Check presence of pluripotency marker.
Is this problem present in the early passages? Sometimes late passages may cause these kinds of problems due to change in their pluripotency.
the problem only occurs after passage 3, cryopreservation in N2 seems to reset it. ROCKi is used following each passage. We have noticed some differentiated cells in the culture, but these largely remain behind following passage. We will be performing some flow analysis soon. Thank-you for everybody's input, it is greatly appreciated- as would any other feedback
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