Problem in culturing MCF-7 cell line!
Posted 28 February 2005 - 11:45 PM
I got some problems in MCF-7 morphology and culturing medium.
My MCF-7 cell line was from ATCC, and its recommended medium is Minimum essential medium (Eagle) with 2 mM L-glutamine and Earle's BSS adjusted to contain 1.5 g/L sodium bicarbonate, 0.1 mM non-essential amino acids and 1 mM sodium pyruvate and supplemented with 0.01 mg/ml bovine insulin, 90%; fetal bovine serum, 10%.
However, I used DMEM to replace MEM. Because some papers signed and I think it still grew well and formed domes as ATCC indicated!Neverthless, its doubling time was not as fast as ATCC indicated.Is DMEM not suitable for MCF-7 cells?
Besides, I observed some phenomena in adding insulin!
When adding insulin in medium though the whole culturing, MCF-7 cells formed domes as clusteres. However, when adding insulin in medium after thawing for three weeks without insulin, MCF-7 cells grew epithelial-like and some domes!The latter way is easy to count cell number. Therefore, should I followed ATCC to add insulin to culture MCF-7 cells?
Thank u for replying!
Posted 01 March 2005 - 01:26 PM
1) MCF7 can be grown in DMEM, but MEM is better.
2) We never normally bother to add insulin for growing MCF7 - just MEM with the extras (FBS, pyruvate etc). Works just fine, and the cells grow quite quickly with an epithelial morphology. Once they get up near confluence, however, loads of them float off - I don't know if this is a consequence of not using the insulin.
So, when you don't add insulin til 3 weeks after you've taken them from frozen, they probably have the morphology mine always do.
I suppose whether you add the insulin or not depends on what you want to do with the cells...
Posted 01 March 2005 - 06:42 PM
doubling time is usually increased to the advertised amount if you put in 10nM estrogen.
they float off in insulin as well, when they're overgrown.
just took some out of liquid nitrogen last week, and they're fine.
Posted 01 March 2005 - 06:53 PM
1)So, you recommend MEM for MCF-7 cells. Do I need to buy another vial cause my MCF-7 cells have been cultivated in DMEM within ten passages?
2)My MCF-7 cells grow more quickly with an epithelial morphology than with domes morphology. And those floating MCF-7 cells may be the result of adding insulin! [Motility response to insulin-like growth factor-I (IGF-I) in MCF-7 cells is associated with IRS-2 activation and integrin expression.
Breast Cancer Res Treat. 2004 Jan;83(2):161-70.] Besides, I got some statements from ATCC product information sheet that most of the clusters remain in suspension until after the 2nd subculture. I found the floating MCF-7 cells alive morphology. However, I didn't collect them in subcultivation. Nevertheless, some MCF-7 cells become floating again! So I think this phenomenon is associated with adding insulin!
3) As far as my research, I exert myself to look for new therapeutics in treating breast cancer. Therefore, I hope what I've screened are meaningful to progress in the future study!
Posted 01 March 2005 - 07:05 PM
I've been growing them in DMEM with 10% FBS, sodium pyruvate, non essential amino acids , 3.7g/L NaHCO3 (but culture in 5% CO2) and 10 microgram/mL insulin. Is the amount of NaHCO3 improper for the growth of MCF-7 cells?
Besides, did u put them in 10nM estrogen to increase the doubling time and how long did they double if without estrogen but with insulin?
Posted 02 March 2005 - 01:03 PM
The media recipie i have for MCF-7 cells is
DMEM to 50mL
FCS (10%) 5mL
Sodium pyruvate (1mM) 500uL
Insulin (10ug/mL) 500uL
Non essential aa (0.1mM) 500uL
Antibiotic/mycotic (1X) 500uL
With 10nM estrogen, there was about 3-4 times more cells than without estrogen. This is from memory, but there was a huge fold increase. Some media recipies recommend adding estrogen. If the media you're using is phenol red free, this also reduces the cell growth rate, because the chemical structure of phenol red is similar to estrogen, and the cells respond accordingly.
Posted 02 March 2005 - 06:09 PM
It sounds like the amount of NaHCO3 in the medium is not the point, is it?
Besides, I add 10 ug/mL insulin in the medium followed by manufacturer's instructions which is different from yours.
Moreover, did u add estrogen and insulin in the medium at the same time?
Incidentally, I used the medium with phenol red and had read associated references as u mentioned.
Thank you for your cherish opinions!
Posted 02 March 2005 - 09:00 PM
using insulin seems to depend on what your working on, and probably habit of the researcher.
i add in 500uL of 10ug/mL insulin into the 50mL final volume. what do you do?
sodium pyruvate is candy to the cells (apparently they can become addicted to it!).
Edited by vetticus3, 02 March 2005 - 09:01 PM.
Posted 04 March 2005 - 07:44 AM
grow my MCF-7 in DMEM 10% FCS, penstrep, fungizone and b-mercaptoethanol. Don't add any insulin or non-essential amino acids.... Do you think this will be a problem?
Posted 04 March 2005 - 10:53 PM
Earle's Balanced Salt Solution and non-essential amino acids
1 mM sodium pyruvate
2 mM L-glutamine
1500 mg sodium bicarbonate/L
supplemented with 10% FBS and antibiotics without estrogen and insulin, and they are happy.
Posted 11 March 2005 - 08:02 AM
Worked absolutely fine.
Posted 25 March 2009 - 03:45 AM
Worked absolutely fine.
Have u added insulin? can u able to send ur protocol on this id email@example.com ? as i have done in RPMI 1640 without insulin but problem
Posted 25 March 2009 - 03:46 AM