I am trying to culture this cell line. Cells do attach to the bottom of the flask but take weeks to grow/divide. Even with that, I wasn't able to get more than 50-60% confluency. My complete media is composed of DMEM, 20% FBS, 1% Pen-Strep and 1% NEAA (changing the FBS percentage from 10-20 and adding NEAA helped a little bit). I also tried changing the culture flasks and realized that the cells prefer growing in one type of flasks. Appreciate any suggestions to optimize the growth rate of these cells.
I have exactely the same problem with theses cells. It is now 3 weeks we cultived them and they did not growth. We actually used DMEM 10% inactivated FBS, 1% pen/strp and 1% Glutamax.
In the litterature I find diffrent protocols for the medium...
- Dulbecco's Modified Eagle's Medium (DMEM) with 1% non-essential amino acids, 2 mM L-glutamine, 1 mM sodium pyruvate, and 1500 mg/L sodium bicarbonate and 10% fetal bovine serum.
- Eagle's Minimal Essential Medium (EMEM) (Sigma, #M0268) containing 10% FBS, supplemented with 1.5 g/L sodium bicarbonate, 1% non-essential amino acids, 1% HEPES, 1% sodium pyruvate, 1% l-glutamine and 1% penicillin/streptomycin.
- Eagle's Minimum Essential Medium, Catalog No. 30-2003. To make the complete growth medium, add the following components to the base medium: fetal bovine serum to a final concentration of 10%.
- Eagle’s minimum essential medium, supplemented with 15% heat-inactivated fetal bovine serum (FBS) (Gibco,UK), 100 IU of penicillin/ml, 100 µg of streptomycin/ml and 2.5 µg of amphotericin/ml)
Have you find a solution to culture fcwf-4 cells, I am very interested to kown your solution Pabasara Weerarathne ?
Thank you for your help!
Océane Floriot , I ended up getting a new vial of Fcwf-4 cells from ATCC (CRL-2787) and followed their protocol.
I prepared complete media using EMEM with 10% non-heat inactivated FBS and 1% pen-strep. My cells did grow well. But it still takes few days for them to reach 80-90% confluency.
Some things that I noticed/learned are;
- these cells are very sensitive
- they start to grow slowly when they are passaged several times (try to use cells that are not passaged too much)
- they grow faster when they are cultured/seeded at a higher concentration (1 x 105 viable cells/cm2). They get pretty laid back when there is a lot of space around them and do not multiply faster
- try not to spin the cells too much. when splitting the cells, I used the exact same protocol given in the ATCC product sheet
Hope this helps.
Pabasara Weerarathne thank you for your answer, I will try your recommandations. We observed also a best growth with higher concentration of cells but I think we have a problem of medium.
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