Once they form small colonies, passage them to an equal or smaller plate area, then they should grow to confluency.

We grow primary fibroblasts from skin biopsies, and those usually take 1-2 weeks to produce colonies large enough to passage. We passage our fibroblasts using this solution: 0.05% trypsin, 500 uM EDTA, and 1% chicken serum. They adhere best if the tissue-culture plate is coated with 0.1% gelatin. 

Hi Anna, 

I have cultived human lung fibroblast cells, with RPMI-1640 supplemented with 10% heat-inactivated fetal bovine serum (FBS), 1% L-glutamine and 50 mg/ml gentamycin. Fibroblasts were derived from lung tissue samples obtained from patients undergoing lung surgery  or video-assisted lung biopsy. The biopsies were immediately placed in cold PBS + 50 mg/ml gentamycin. Then, biopsies were finely minced, washed twice with cold PBS + 50 mg/ml gentamycin and then plated in petri dish., before plating minced tissue, petri dishes were pre-coated with heat-inactivated FBS (a volume suitable to cover the entire surface of the plate) and allowed to dry in a sterile hood. Of note, pre-coated plates were prepared in advance to tissue sampling. After planting the plates should be left in the incubator for at least 48 hours without being moved tro allo

After seeding, petri dishes must be left in the incubator for at least 48 hours without being moved, to allow small pieces of tissue to adhere to the plate.

After about 7-8 days, at the base of each tissue fragment will appear fibroblasts.

One important tip is that the primary cell lines should be splitted up to 1: 3 or 1: 4, and never have to go to the confluence otherwise they will stop to proliferate.

Good luck 

Loredana

I will agree with Loredana and would suggest you to follow her protocol properly. I do similarly for my lens primary culture as well as corneal fibroblast culture.

Thank You very much for many information. I'll try to implement them.