cell splitting problem

[kjell madsen]

<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
>
>michael baron

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.

Kjell Madsen