Hi,
I am facing problem to grow fibroblast
1) fibroblast growth is very slow
2) preserved cells grow abouth 60% after thawing
3) the growth of fibroblast cells achieved after 8-10 days
4) the cells does not adhere to the plate properly, so which kind of coated plate I should use
I need help and suggestions to resolve these problem
Thanks
We use gelatin-coated tissue-culture dishes for primary fibroblasts derived from patient biopsies. We also start to see colonies after about 10 days.
What kind of fibroblasts are you growing, and how do you dissociate the tissue you are deriving the fibroblasts from?
I use tissue culture dishes and we have no problem to obtain fibroblasts. Check that your growth medium is suitable for fibroblasts and calcultate your doubling time. Good luck
It may help to seed your fibroblasts at a higher density. Especially when you thaw them, fibroblasts like to be thawed at a higher density than you would normally use when passaging them.
Benjamin please guid me for the fibroblast medium composition please than i can match with mine medium composition
Juia your suggestion to use gelatin coated plates is really good and normally fibroblast grow with in 5-6 days and you see colony appearance after 10 days. Do you think after ten days they will respond good if any treatment is applied
For passaging primary fibroblasts I use a seeding density of 1x10^4 cells/cm^2. In other words this means 750000 cells into one T75 flask.
For thawing primary fibroblasts I use a seeding density of 4x10^4 cells/cm^2. In other words this means 3000000 cells into one T75 flask.
I hope this helps!
Hi Imran,
Once the primary fibroblasts form colonies (about 10 days), we passage them with attenuated trypsin. Usually we do a "reverse split," where we pool several wells containing small colonies together into a single well. For example, we might have 3-4 wells on a 6-well plate containing 2-3 colonies each, and we would split those onto 1-2 wells of a new 6-well plate. Fibroblasts grow best when they can touch each other. I wouldn't recommend doing any experiments with your primary fibroblasts before passaging them at least once.
Good luck!
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