Protocol Online logo
Top : New Forum Archives (2009-): : Tissue and Cell Culture

number of splitting after defrozing cells - (Aug/29/2012 )

Hi, sorry for infantile question, but I am wondering after defreezing cells how many time they should be splited before use them to investigation ? It is up to cell line type, conditions ?

Thank you for sharing your experience in advance,



Preferably the minimum number you can to get the amount of cells you need. The longer you have a cell line up, the more it changes from the parent line (sort of lab evolution), so that in the end you may be working with a cell line that isn't actually what you think it is. This is one of the reasons that people are being asked to validate their cell lines before papers will be accepted at certain journals.


Hi Magi.
In addition to what bob1 said, you need to check the regular morphology and preferably the parent culture's growth characteristics (doubling time etc) in the new culture. What we do for most of our cells is as follows: after thawing the cells, we start the culture into a T25 or a T75 flask, then try to observe the expected morphology until cells reach 50-70% confluency. Then do the trypsinization+cell count and start the assays with the desired number of cells. Therefore it is only one more splitting (assuming you can get enough number of cells from that T25 or T75). For the morphology of established cell lines you can check ATCC web page, they provide pictures of low confluency and high confluency pictures of the cell lines.
hope this helps.