Our group is working on breast cancer tissue doing cell culture, preparing for a future stem cell isolation based on cell markers.
We could see too many cells floating in the media on the day of dissociation (with Collagenase 4 ), we did not get any cells attached to the flask on the second day of dissociation.
We mince about 1gm of tumour in 10 ml collagenase (1mg/ml, contains s/p, fungizone and glucose) and incubate for 3 hours. Then we spin for 4 minutes at 4000 rpm to collect the precipitate. We then resuspend the precipitate in 10 ml complete media containing fungizone and s/p overnight.
We are following a paper published in this field (long-term cultures of stem/progenitor cells from lobular and ductal breast carcinomas under nonadherent conditions).
Note that: in this paper, 6 growth factors are used, but we are waiting for it to be delivered to Egypt.
Also 2 types of media are used and we use only one ( Ham's f12 ) with FBS up to 40% (Active serum)
Do you have any idea why I have this problem?
Is this trouble due to the absence of growth factors?
Or, because of using only one type of media?
Or, the FBS should be heat inactivated?
Or should I use another protocol for dissociation and primary culturing?
Salma