fibroblast contamination in primary culture - (Nov/18/2004 )
I am trying to stablish primary cell cultures from lung tumour specimens, but fibroblast overgrow in my cultures, and epithelial cells are not able to grow properly. Can anybody help me?
If you use a special media for keratinocytes witout serum, the fibroblast should not outgrow your epithelial cells. A basal media with high osmolarity (340 m Osm/kg) with high level of aminoacids, Epidermal growth factor 1,6 nM, Hydrocortisone 1,4 µM, Ethanolamine 100 µM, Phosphoethanolamine 100 µM, Insulin 150 mU/ml, Bovine pituitary extract 20 µg protein/ml.
You can also buy Keratninocyte serum free media from Invitrogen.
Primary epithelial cells grow better on fibronectin/collagen coating:
100 ml Basal medium
10 ml BSA (1 mg/ml)
1 ml Collagen I, bovine (2.9 mg/ml) (BD Laboratories #40231)
1 ml Human fibronectin (1 mg/ml) (BD Laboratories #40008A)
To coat T-25 flasks with fibronectin/collagen/BSA coating solution:
Add 2 ml of coating solution to a T-25 flask. Stack your supply of coated flasks on a flat tray. Transfer the tray to an incubator and allow the coating solution to sit for a minimum of two hours or overnight.
Take the tray out of the incubator and remove the excessive coating solution from each flask. Flasks should be allowed to dry flat in the hood for at least one hour. Coated flasks are stored at room temperature in their original bags. Coated plasticware is generally stored for <1 month because of our high usage. A similar procedure is followed for preparing all culture flasks and culture dishes for growing our epithelial cell lines.