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Cell culture of MYC immortalised HDFa - need advice

media composition cell culture

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#1 That_Lab_Guy

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Posted 18 March 2014 - 10:29 PM

Hi everyone.

 

I am relatively new to cell culture and want to find out how other researchers/labs culture dermal fibroblasts, typically MYC-immortalised HDFa (adult human dermal fibroblast).  However, if the derivation of the cells are not from MYC immortalisation, you are also welcome to share your method of cell culture with regards to media composition.

 

So I have been using DMEM (http://www.lifetechn...ulation.14.html) supplemented with FBS  (ES Cell Qualified Fetal Bovine Serum), Sodium pyruvate (Sodium Pyruvate 100 mM (100X), liquid), PSG (http://tools.lifetec...50571_10378.pdf) and NEAA (MEM Non-Essential Amino Acids Solution

100X).  All from Life Technologies.
 
The volume and final % concentrations of the supplemented medium are as follows:
 
DMEM - 435 ml (87%)
PSG - 5ml (1%)
NEAA - 5ml (1%)
Sodium pyruvate - 5ml (1%)
FBS - 50ml (10%)
 
However, I realised that my cells are growing a little too slowly so I decided to try a few other medium types such as Medium 106 supplemented with LSGS also from Life Technologies which I supposed should be more favourable for culturing dermal fibroblasts as stated on their website.
 
I also prepared other customised supplemented media such as these:
 
DMEM - 47ml (approx.)
PSG - 500 ul
NEAA - 500 ul
Sodium pyruvate - 500 ul
FBS - 1 ml (2%)
 
DMEM - 47ml (approx.)
PSG - 500 ul
NEAA - 500 ul
Sodium pyruvate - 500 ul
FBS - 1 ml (2%)
EGF - 5 ul (10ng/ml)
Dexamethasone - 6.45 ul (1uM)
Heparin - 50 ul (10ug/ml) (http://www.sigmaaldr...ng=en&region=SG)
 
*concentrations shown are in final
 
I don't know if there are any problems with these media compositions.  But if there are, I would be happy to hear from you all.
 
As of now it is still day 1 of trying out these 4 supplemented media types so it is too early to tell which is best suited to grow these cells.  That being said I would like to find out from anyone out there.  How exactly do you culture dermal fibroblasts?  I am only looking at immortalised cell lines, not primary culture. What is your culture media composition and if possible can you explain why you have used such a composition?  Also I have tried searching for journal articles online of how other researchers culture dermal fibroblasts but I still can't really find any.  Please do share with me any journal articles and their URL links emphasizing on the cell culture media composition under Materials and Methods.  Also, is there any difference in media composition for the culturing of NHDFa and immortalised HFDa?
 
 
THANK YOU AND MUCH APPRECIATED! smile.png

Edited by That_Lab_Guy, 18 March 2014 - 10:41 PM.


#2 bob1

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Posted 19 March 2014 - 01:25 AM

The only major problem that I can see is your extensive use of antibiotics.  If your culture techniques are good enough you shouldn't need them at all, and they should never be used routinely in culture as they can hide low level antibiotic resistant infections that alter your cells in a similar manner to mycoplasma.

 

The only times you should need to use antibiotics are when you are making a stable cell line that requires selection for a particular marker (in which case the antibiotic will be a eukaryotic one such as geneticin) , or if you are culturing a primary cells that could be contaminated with bacteria, such as gut cells or skin cells, but even these you should discontinue the antibiotics once they are a few passages in.







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