Our lab is finding it difficult to expand MSC's . When transferred from a T-25 flask to a T-75 we observe a marked reduction in their proliferation. They do adhere to the flask but fail to proliferate. Does it have something to do with the seeding density ? We subculture them when they achieve 80-90% confluency in T-25 flasks.
Do your cells grow well in T25 flasks? Are the T75 flasks from the same brand/have the same type of filter? I had problems growing a normal lung cell line in flasks because they need more oxygen than regular cell lines. The solution was to grow them in petri plates.
hi mafalda , yeah both flask types are from the same brand and have filtered caps. Just like you, to circumvent the problem we have switched to petri plates. they grow well in a petri plate but not in a T-75 , i don't understand why.
Hi Shashank, in brief I do know why you are having problems, I routinely passage hMSC in T175 flasks (seeded at 7x10e5 per flask). The hMSC I use were isolated from bone marrow aspirates (not sure if source may have an effect of oxygen requirements etc?). The cells grow at 5% CO2, 37oC.
I was wondering are the caps of the flasks that you use are different between the T25 and T75? We use vented caps in our flasks but you may want to check just in case you got some un-vented (which need to be loosened before putting in incubator).
Sorry I can't be more help,
Gary
hi gary thanks for your reply . Getting my MSC's to grow in a T-175 , like you , looks like a distant dream at least for now. Both the flask types do have vented caps and i use the same culture conditions . I will try seeding them at a higher density.
Hi you can try that though I don't believe density is a problem (they are selected as colonies from MNC and I seed a T175 with quite a low density). Could it be the passage level you are working at? Higher passage MSC will eventually stop expanding (HayFlick/senescence). The failure to expand after seeding is what I have seen with ps. 8-10 cells (after passaging from ps.2 in T175 flasks at the density above).
Anyway, good luck!
hi gary thanks for your reply . Its passage 3 which i am trying to expand
is the media volume which i am using during culture the problem. I culture my MSC's in a T-75 flask containing 5 ml media . Does this effect diffusion of gases.?
Hi Shashank it does seem a bit low. I routinely use 10-20 ml for a T75 and 30 ml for a T175.
What FBS are you using? If you can passage in T25 it is unlikely to be the FBS but the quality of the FBS is pretty critical for some cell types. Gibco and ATCC both produce pretty top grade FBS so I would use either of these. You might also want to try different FBS types to see if this makes a difference.
For my hMSC (which grow well) I use DMEM (Gibco) + 10% FBS (Gibco).
Hi Shashank,
Not sure if you're still having difficulties, but we've found that the MSCs grow better in Falcon 3003 tissue culture dishes. Growth in the T-25 flasks is similar (albeit the cells look more stressed), but when scaled up in flasks, the cells slow down.
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