MCF-7 cells clumped and too small - (Dec/15/2006 )

I wish I had a microscope with a camera so I could show pictures, but I'll give my best description. My "old" MCF-7 cells that I have been using for about 4 months have always looked like this:
http://www.cf.ac.uk/phrmy/Tenovus/web_pics...cs/WT-MCF-7.jpg
(sorry for the small size).

I had problems with contamination, so I started another "new" line from a *different* lab's frozen stocks instead of my own (just in case my stocks were contaminated, which it turns out they weren't). Now they grow approximately 10 times smaller than my originals and ONLY in clumps (sorry no photo available - I could draw one?). They will never spread to fill the plate like my "old" cells do. They are treated the exact same way, except lately I have been attempting to mix and/or trypsinize them more thoroughly (and double checked under microscope that I have single cells, no clumps), but that still failed to "fix" the clumping. Other lab members ensure me they ARE MCF-7s, just seem to grow funny.

I called ATCC and they recommended passaging them a few times and it may go away - it didn't. They also claim they've never tested the type of media I use (MEMalpha w/ phenol red, 10% FBS, glutamine, pyruvate, non essential amino acids, pen/strep) but it works beautifully for my "old" cell line. They also claim they never use media without insulin. Another interesting point is that they were shocked to hear I resuspend cells with a 1mL pipet tip or sometimes pipet with a 200uL pipet tip. They say they never use anything less than a 2mL pipet or else you could break cells, release DNA and cause clumping. (To subculture, I wash, trypsinize TrpLE solution 1mL for a few minutes, suspend in 3 more mLs media, spin, resuspend with 1mL tip and aliquot with 200uL tip). I doubt this is DNA-induced clumping as both lines are treated exactly the same way and they have been passaged many times.

I could just give up on the "new" cell line since my old was not contaminated and I use it currently. However, I am so intent on finding out why the new cells are doing this that I am keeping them going. I may try letting them grow for as long as possible to see if they EVER fill the dish, but it seems they will only stay in those clumps with a ton of free space around. I may try adding DNase to the media to confirm my suspicion that it is not DNA induced clumping.

Has anyone heard of this or have any ideas what is going on?

Something similar has happened to me. I had MCF7 that I was working with in late 2005, got some nice data, but soon had trouble thawing my freezes previously made. Bought new MCF7 from ATCC, and they grew so slowly and took forever to cover the dish, like a month, I called them they said this lot grows weird, in 3D clusters and it takes a long time to become confluent at first (2 weeks they said) I tried the media they recommended but what was happening was they wouldn't attach well. Tried another vial from ATCC, same thing, until I passed them a few times to "select" for the good attachers. However, now they grow, still slow, but I can't repeat the results I got in late 2005 so I can't go on with the project (well, I will keep trying) I'm hoping the "older" these cells get they may become similar to my previous batch?

Maybe if you pass them a few times they will improve?

Those weird cells were from another lab's frozen stocks. We were using them because I thought even my stocks might have been contaminated. It turns out, though, it was just too many people using my incubator and the incubator was contaminated. I went back and used my own. So, I went back to using my own frozen stocks and they looked like normal and I had no major contamination problems since.

I did talk to ATCC over the phone and they said if you break the cells and DNA spills out, all the cells will clump on that spot so it could be a problem of DNA release, too. I have never had that problem before though, so I am doubtful that is it. My other suggestion is to go back to the earliest stocks possible and check all your frozen stocks, throw out the ones that clump like that, if possible. Check with labs nearby to see if they have any. If you absolutely can't get more cells, you should check the genes and make sure they are MCF-7's. That's what I was going to do. We should also try to come up with a selection method for picking only healthy cells. Perhaps dilution series, grow up in 96-well plates and look for the best growers or manually pick-up regions of healthy looking cells and transfer to new dishes.

I would complain to your supplier if I were you and get them to send you new cells instead, too.

Hi

I work with MCF7 too but i grow them in RPMI media. I had similar problems too. I have frozen stock that i made my slef and i noticed differences upon thowing cells from tube to tube. sometimes they are small and attach very well to the dish and one time they were big and and sick. my experiemts usually work well when the cells are small and grow in clumbs..

thanks

Hi

I work with MCF7 too but i grow them in RPMI media. I had similar problems too. I have frozen stock that i made my slef and i noticed differences upon thowing cells from tube to tube. sometimes they are small and attach very well to the dish and one time they were big and and sick. my experiemts usually work well when the cells are small and grow in clumbs..

thanks

Hi,
I used to work with MCF-7 from ATCC. I grow them in RPMI supplementing with 10% FBS, NEAA, pyruvate, glutamine and without phenol red. They never give any trouble to me and grow quiet fast. I never come across any clumping MCF-7. Maybe it was the stock?

Regards,
cattoyee

I start working with MCF-7 cells, and they clump also. When subculture, I aspirate medium, add 2 ml Trypsin/EDTA and incubate at 37oC for 3-5 min, then resuspend cells with 8 ml medium by pipetting several times. However the cells til clump and it seem not good for subculture. Some1 suggested me pipette more until no clump as seen in the microscope or using syringe in order increase pressure.
My question is that so many time pipetting or high pressure through syringe can affect on cells, maybe cell membrane breakdown or DNA out??

Thanks for kind replies.