11 replies to this topic

[refolder]

Hi all,


I want to scale up my U-937 suspension cell line. My problem is, that the cells sink to the bottom of the flask, grow to confluency (like adherent cells, but they dont attach to the surface), and that point their division ends.

Should I use a shaker to my U-937 to produce more cells? Or simply escalate the growing surface? Any other idea?



Thanks for any reaction

[gfischer]

refolder, on Nov 13 2009, 08:49 AM, said:

Hi all,


I want to scale up my U-937 suspension cell line. My problem is, that the cells sink to the bottom of the flask, grow to confluency (like adherent cells, but they dont attach to the surface), and that point their division ends.

Should I use a shaker to my U-937 to produce more cells? Or simply escalate the growing surface? Any other idea?



Thanks for any reaction

You could use a larger flask, or just split the cells into multiple flasks. I'd recommend against the shaker; the agitation may aerate the medium, changing the pH.

[bob1]

Roller bottles may work, though they are mostly used for attached cells.

[DRT]

I’m not sure this will help but I occasionally use U937’s and they always stay in suspension until I differentiate them with PMA. I use 10 ml cultures and split c.a. 1/20 three times a week because they can get finicky about their density. Are you using the RPMI with 10mM HEPES that ATCC recommends?

[refolder]

Unfortunately the roller bottles are too expensive for me, but good idea, thanks.

I'll try what gfischer said.



DRT:
May be the problem is that I use NaHCO3 not HEPES?
I also use PMA but with 0,2% lactalbumin hydrolysate, and the cells stay in suspension.

[SuMi]

Why do you use PMA? I only use it when I want to differentiate the U937 cells into macrophage-like cells and it makes them adherent. Otherwise my cells grow in suspension

[refolder]

SuMi,

When the U-937 cells are induced with PMA, they produce MMP-9, and that's what I need.
Do you add serum to the cells when you differentiate them? When I once left out the serum, they adhere to the flask, without PMA induction.

[SuMi]

Yes I always use serum. Could you lower the dose of PMA to avoid differentiation while still stimulating MMP-9 production? What concentration are you using? Other than that I have no ideas, sorry!

[refolder]

I use only 50 ng/ml PMA.

[SuMi]

That's the concentration that I use to differentiate them into macrophages so that explains why they are adherent!

[Rayme]

I am planing to work with U937 cells. I want to use PMA to differentiate. Could you please tell me the best concn of PMA...ng/mL for the differentiation? How fresh PMA do we need? do we need to prepare every time we use or we can keep in refrigerator and use for 2/3 months? I would appreciate any kind of help regarding the culture and differentiation of this cell line. thank you

[eck]

I've just started using PMA for my U937 cells. They tend to clump and some might adhere if you add about 10 nM of PMA. They are quite easily detachable - just a bang on the flask will get it off.
Some journals I read use 10-100 nM range.

I'm not too sure about the extent of how fresh your PMA needs to be, but assuming it's stored in freezer and dissolved in DMSO, it should be alright.
You should ideally use a new frozen aliquot every time you want to differentiate these cells.

I'm not sure if it's just me, but I realised that these cells tend to stop growing after you add PMA. It might be different under the hands of others, but that's just what I've realised.

Edited by eck, 16 June 2011 - 02:49 AM.