I recently switched from Human Recombinant Insulin to Insulin from bovine pancreas and my MCF-10A cells start to look weird after a few days in culture. They don't become 100% confluent, tend to be patchy and not the characteristic "cobble-stone" like appearance that they should have. Anyone know if this could be a result of the change of insulin or any other ideas?
I would imagine given that this is the only change you have made that it is your new insulin (I assume that this is the only change you have made, and all the other additives are the same eg serum, cholera toxin etc). Have you tried growing two sets of cells in parallel, one with the old recombinant insulin, one with the new bovine insulin, or "rescuing" your cells grown in the bovine insulin with recombinant insulin?
Hi "Andrews", as A Sunters said, it looks like that's the reason. We've been working without any problem with Sigma's recombinant Insulin http://www.sigmaaldrich.com/catalog/product/sigma/i9278. Alternatively, we've used Roche's recombinant Insulin http://www.roche-applied-science.com/proddata/gpip/3_5_3_10_6_2.html. Unfortunately, I've never worked with insulin from bovine pancreas so I cannot assure that's the problem. Good luck!
MCF10A cells are very sensitive to changes in culture conditions, and, when not optimal, they can lose very rapidly their epithelial properties ( as their ability to form acini on matrigel). It indeed seems that the insulin might be the problem in this case (yet I have never used bovine insulin myself), but one of the most common causes for this type of behavior is cell crowding. If you have been keeping your cells at high cell densities (even once or twice), this will be enough to ruin your cells. MCF10A cells should NEVER reach high confluence in their flasks.
Thank you guys for all the great answers.
A Sunters: I am going to try to grow them with the different types of insulin side by side. I also made a new batch of media with new ingredients to check if my old media might have gotten contaminated.
Mariano: We used to use human recombinant insulin form I think sigma and am going to order it again.
Ivan: Thanks for that tip. I actually noticed this after I thawed a brand new vial that was given to our lab from a lab that specializes in working with MCF-10A cells. Interestingly, after I thawed the cells, plated them and looked at them the next day, they looked like "normal" MCF-10A cells. Then after a few days in culture they started to look weird. This problem didn't happen to me before, as I've been using MCF-10A cells for a few months now.
Anyways, I'm going to try out a few things and hopefully figure out whats going on. Thanks for the help.
In principle, if the quality of the bovine insulin is highly purified and suitable for cell culture this should not be a problem.
To identify the problem, you need to find out whether the culturing or the start up from your ampul is problematic or whether the bovine insuline used is the cause.
If you still have medium with recombinant human insuline you can split the cells and give either supplementation with bovine insuline or recombinant human insuline. Find out which are growing better. Second choice is start up to ampuls with both insuline from human and bovine origin. If bovine is causing the trouble, try other bovine insuline preparations. Normally it can be used without any problem..
Hello,
I think also that it may be a problem relatred to the Insulin from bovine pancreas. It has an animal derived origin and it may contain mycoplasma, adventious viruses,... It is better to return to Human Recombinant Insulin. To be sure you can do a comparative study.
good luck!
^ Yes, I'm using the correct recipe. According to Denath et al 2003. At the moment I borrowed some media from another lab whose MCF10A cells are looking good and am comparing my media with theirs side by side using a new vial of MCF10A cells. We shall see! Thanks again for your responses
I have the same problem now with my MCF-10a. Before this month, My MCF-10a was working well. but I don't know why it looks weird now. And I try to thaw a brand new vial, the problem is still there. I do not know if you have solved this problems？Could you give me any advice?
I changed a few things in my media but I don't think it was the insulin because it should work with both human and bovine insulin. Try making all your ingredients fresh and see if you can get a new vial from another lab that is at a low passage number. This worked for me. Hope this helps.
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