what protease is best for primary cell culture - (Aug/06/2008 )

hi all, hope you can help.

I am currently doing a PhD in ireland and am quite new to the area of cell culture especially primary cell culture.

the question i have is that when i try to detach my primary granulosa cells from the (35mm) tissue culture dish they are growing on, they always have trouble fully detaching.

If i watch them under the microscope after adding the trypsin/EDTA i can see the cells getting smaller as the retract or lose their attchments. I see the cells "ball up" although i am sure this is not the correct term, (basically they form spheres with no apparent attachements to the plastic)

However when at this stage any cell line i have worked with before would float with a little tapping or shaking the primary cells remained attached to the bottom of the dish and will not remove even with some hard taps on the bench

I have even left them for 20 minutes or so in the trypsin just to see if they would remove but they dont

One funny thing is though is that when i add medium to the flask to stop the effects of the trysin some cells(about 10 % of overall number) now detach from the dish and when they are placed into a new flask they are viable. (i think this could be the action of the media washing against them but maybe i am wrong)

so does anyone have any advice on what protease to use for the detachment or exactly why these primary cells do not detach fully even when they appear to be detached?

any help would be great

Colm.

First, are you washing your cells with magnesium- and calcium-free DPBS? Adhesion proteins require divalent cations to work, and rinsing them in PBS without divalent cations helps cells to detach.

Secondly, the cells won't all detach by themselves. Unless they're very stubborn, though, they'll come off when you add media+FBS to neutralize the trypsin. You do this by rinsing the inside of the flask with the media when you add it.