how to culture a cell line of "fibroblast" - (May/20/2002 )
how could i find the associated data about culture "fibroblast"?? for example :which organ i can use, the medium,the factors affecting culture..and so on
can sombody tell me? thank you very much!!
depends what kind of fibroblasts from which organism you want to culture. If you do not need to establish a primary culture, contact a tissue culture bank (like DSM in Germany).
i receive your message, i think it will be helpful to my research . thank you very much!!
if u want to use Fibroblast cell line, try NIH 3T3 cells but u can also use skin for primary cells culture.
I make primary cultures of fibroblasts from gingiva. I use a mix of DMEM and Ham's F12 supplemented with 10 % FBS, pen/strep, L-glutamine. I don't add any particular supplement. These cells adhere to the flask easily. I suggest a minimum seeding density of 50 %. I use trypsin/EDTA to do the passages.
You can get fibroblasts from tissue by outgrowth. Cut your tissue samples in about 2mm² squares and let them dry to a 25cm² flask (about 5-7 pieces per flask) for about 30 minutes. Add cell culture medium (I use an equal mixture of DMEM and RMPI with 10% 1M HEPES-Buffer/ 10% FCS/ Penicillin /Streptomycin) and culture them at 37°C/ 5%CO². After one to four weeks you will see that cells grow around the tissue pieces.
You have to passage the cells when they become too close. The pieces of tissue can be discarded with the first passage.
Careful with skin samples! Ceratinocytes have also the ability to outgrow. Put the pieces in the flask so that the epidermis has no contact to the plastic. But even if ceratinocytes grow in your first culture usually you will remove them with the passages, since they get not detached within 4-6 minutes like fibroblasts.