Hello! I am trying to culture hTERT-modified fibroblasts, derived from human fallopian tube tissue. I am looking to run a flow panel on the cells, and so I need a good amount of them to get accurate data. However, the cells grow so slow. Currently, we are culturing them in:
1/2 Pri-grow media
1/2 F12 media
10% USG
1% Anti-Anti
+ 5ng/mL bFGF
The bFGF is added fresh to the media each time I use it. I suppose I could go up in concentration, but right now I am just following the protocol from previous lab personnel. Another researcher suggested that maybe doing partial (half) media changes as opposed to full would be helpful. Would this help my cells grow faster? Is there anything else I could be doing to improve their culture conditions? Thanks!
Note: We are culturing the fibroblasts in the same base media that we culture our FTE cells in (without the bFGF) for a series of coculture experiments we are running. So if possible, I would rather not alter the media recipe too too much.
Dear Lottie
Often fibroblast do not pose problem in growth when it has been already immortalised with hTert.
Partial media change certainly has an impact in cell growth and proliferation. This is one thing that you can definitely try.
In addition to this you may try using high glucose media, adding growth supplement like glutamine, pyruvate (provided these supplements do not interfere with your assays).
You may also try using antibiotic free media.
Regards
You can consider next improvements for your protocol to enhance growth rate:
1. Remove anti-anti from culture media and keep your aseptic technique at high level.
2. Change cell culture plastic – some plastic treatment not good enough for fibroblasts growth.
3. Adsorb collagen to cell culture plastic to give more attachment motifs.
4. Check your bFGF shelf life: 1 week at 2..8C, 1 year at -20..-80C. Reconstitute bFGF in 0.1% carrier protein (BSA). I recommend store at -80C small aliquots and change aliquots each week.
5. Minimize opening and closing of incubator if your incubators are old – temp and CO2 fluctuations can affect them too.
6. Optimize cell seeding density and confluence. Depends on cells try to subculture fibroblasts at confluence 60-70% – once you see that fibroblasts start to touch each other and form stable connections. At this time they got growth contact inhibition. If keeping them long with contact inhibition later it will take them longer time to restore their division.
Hope it can help you. Feel free to contact me if you have any questions.
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