cell splitting problem
kjell.madsen at upp.pharmacia.se
Wed Mar 13 12:28:31 EST 1996
<Unknown> (morbillivirus group) wrote:
>I have a problem passaging some chick fibroblast (OU2) cells. When I use
>the recommended mix of trypsin/edta, the cells come off as a single sheet
>that does not want to split up, even if the cells are not yet confluent
>(though with lots of intercell connections). Anyone ever experienced this
>as a problem with any other cells, and overcome it? If so, how?
>thanks for any help you can give
Here are some suggestions:
1) If splitting has worked before with this cell type (or you have
obtained the method from a paper), the prime suspect is the tryspsin
solution. Try a new batch.
2) A change in technique may help. One trick is to just rinse the cells
with trypsin/EDTA leaving only a thin liquid film. The cells are then
kept in place by gravity but the trysin/EDTA in the film is usually still
enough to detach them. Incubate for 5-10 minutes at 37C and watch the
cells in the microscope. When the cells start to become rounded, give the
flask a sharp tap on the side.
3) Some cell types produce a lot of extracellular matrix and they can be
very diffucult to disperse. ECM acts as a glue. It sometimes helps to
first digest it with pure trypsin or collagenase, adding the EDTA later.
More information about the Cellbiol