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Cell clumping during thawing - (Aug/19/2005 )

Myself and other members of my group have been having a lot of trouble with clumping of cells after thawing. Cells are thawed by usual method and then gently added drop by drop to warmed media. Cells are then transferred to a flask and left overnight before media is replaced. Our cells seem to clump at all stages - during addition to medium, after centrifugation (when performed) and even in the flask overnight! We have tried adding warm medium to the cells and vice versa, not centrifuging, added DNAse (!) and nothing seems to work! Has anyone else had this problem? Any suggestions?



I don't know what cell line are you working with, but I am working with HepG2 and it is really really clumpy one. I´m not surely the most experienced, but I have been adviced to just mechanically disgregate them by pipetting when I split them, and that is working fine.



many cells can form agregates after thaing if they are able to establish connections between each others. Moreover, freezing treatment can disturb gently the membrane and drive a clumping. The problm is solved after the first trypsinisation for adherent cells and by pipetting carefully for non adherent cells.