One of our PhD stutdent got a problem, he is trying to culture Condrocytes, but the cells are growing very well in one media(D8900) from sigma, but the another media (D6421) from sigma again which he is trying to grow, the cells are not happy not proliferating at all. But the medias are basically similar by the composition, and no other ingredients are different, but the cells from the same sample behaves different, anyone have any cue what and why it could be, many thanks in advance.
senthil;-)
The D6421 lacks L-glutamine, which is often a requirement for cell growth. If the media has not had L-glutamine ADDED manually, or the L-glutamine stock used was old, the cells will grow very slowly if at all. Their morphology is also likely to be abnormal. The D6421 also has sodium bicarbonate, which the D8900 lacks, which may be another factor. However, I'd try supplementing with L-glutamine first.
Thanks for writing Steel, Forgot to mention we supplemented the medai with both L-glutamine and Sodium Bi carbonate. Still the problem persists.
Thanks for writing, and sorry for delay.
I cannot understand what is decomplemented means??!!
But the answer for the next question is Yes, in the same CO2 incubator one flask cells were growing other one isn't, this is ofcourse the problem with the media but where thats what I cannot understand.
De-complemented serum usually means heat-inactivated. We incubated ours at 60 degrees C for approximately 20-30 minutes to achieve this. Other than that, L-glutamine is not very stable; if the stock is more than one week old (or has been thawed that long) I would suggest making fresh stock. Note that you should NOT be adding sodium bicarbonate to the D6421 since it already contains some.
Check this one on the effect of sodium bicarbonate on growth/morphology of chondrocytes: "zwitterion buffers (e.g., HEPES) are used in culture media because they can perform under both slightly acidic and alkaline conditions. However, because chondrocytes tend to rapidly acidify the media, better pH maintenance may be achieved by utilizing additional buffering agents that operate under acidic conditions (e.g., bicarbonate). The purpose of this study was to investigate the combined effect of HEPES and sodium bicarbonate (NaHCO(3)) on extracellular pH and matrix accumulation by cultured articular chondrocytes" Tissue Eng. 2004 Nov-Dec;10(11-12):1633-40.
Effect of sodium bicarbonate on extracellular pH, matrix accumulation, and morphology of cultured articular chondrocytes.
Waldman SD, Couto DC, Omelon SJ, Kandel RA. http://www.ncbi.nlm.nih.gov/pubmed/15684672
In addition, check this two chondrocyte cell lines for propagation conditions at ATCC:
CHON-002- http://www.atcc.org/ATCCAdvancedCatalogSearch/ProductDetails/tabid/452/Default.aspx?ATCCNum=CRL-2847&Template=cellBiology#aProp26c45
CHON-001 http://www.atcc.org/ATCCAdvancedCatalogSearch/ProductDetails/tabid/452/Default.aspx?ATCCNum=CRL-2847&Template=cellBiology#aProp26c45
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