I am culturing primary cells and use DMEM F12 (1:1) cell culture medium supplemented with human insulin and EGF. How long are these components stable for in the medium?
Thank you for your gentle attention.
Regards,
Celina
Hi Celina,
I have used DMEM F12 supplemented with basic FGF and EGF, so a little different, but after the 2nd to 3rd week the media lost its efficacy. While I am not sure how long insulin can last in the media, my experience was that our media had to be thrown away at the three week mark. Hope this helps.
-Daniel
1) A study from Sigma on stability of insulin and IGF-1 in cell culture:
https://www.sigmaaldrich.com/technical-documents/articles/biofiles/long-r3igf-i-an-insulin.html
" Insulin is degraded in cell culture by enzymes (insulinases) secreted by cells into culture media. In addition, insulin is more rapidly internalized and degraded compared with IGF-I and LONG®R3IGF-I. The extended cell culture stability of LONG®R3IGF-I results in prolonged activity and associated benefits to cell culture. (Graph 5)."
In their conditions, 50% degradation of insulin during cell culture occured after 1.5 days.
2) I would supplement it further with trace elements like selenium as it was done in Richard Ham's group in further versions [MCDB] of F12 media (this way the media will provide all the necessary nutrients/elements - though, the concentrations won't be necessarily optimum):
Chapter Survival and Growth Requirements of Nontransformed Cells
Article Growth and Maintenance of HeLa Cells in Serum-Free Medium Su...
3) If you don't add any protein to the media (e.g. 0.1 mg/ml BSA or some more indifferent protein), there may be a problem with adsorption and inactivation of the tiny amounts of growth factors on plate (or other vessel) walls. That's a real problem when you dilute enzymes - e.g. luciferases - to very low concentrations (or look at proteins released into a serum- and protein-free culture media by cells): Article How to prevent losses of protein by adsorption to glass and plastic
(Eg when looking at enzyme activity immediately after a dilution into PBS buffer, 20-100 ug/ml BSA or lysozyme prevents activity loss while 2 ug/ml offers poor protection)
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