WEHI-3BD cell culturing - Advice on culturing. (Jun/11/2011 )

Hi all,

I have a question in regards to WEHI-3BD. In the past, our lab has used RPMI + 10% FCS, 1% PS and 1% Glutamax to culture these cells for IL-3 production.

This year however, we've chosen to use the method as described by Galli's lab (http://gallilab.stanford.edu/protocols/documents/2WEHI.pdf), which they use DMEM media. But it seems the cells don't seem to grow as well, and somehow they appear much more adherent than usual. It gets to a point where I need to constantly blow the cells using pipettes or taping the flask. And even so they just won't come off. We try to avoid using Trypsin to get them off. The other option I've tried is using PBS to wash the cells, and that seems to work. But we're not sure if that causes the cells to die.

Does anyone have suggestions? I just don't know why the cells are not growing. I'm inclined to believe it's the media.

This is what I've done so far. I took them out of cryofreeze on Wednesday. I saw the cells and they looked rather confluent on Thursday, which is why I've moved them to a T-75 flask. But on Friday, I moved some cells from T-75 to T-175 flasks. I checked on Sunday, they weren't growing. Even in the initial T-75 flask, which I did about a 1 in 2 split.

Thanks,
eck.

Did you wean them onto the DMEM or just go straight from RPMI to DMEM (freezing doesn't count you still need to wean)... if not, then this may well be your problem. To wean, you should slowly change the cells onto the new medium by adding differing proportions of DMEM:RPMI over several passages, starting with a low amount of DMEM (say 10-20%) and working your way up to 100%.

Interesting. I just moved them straight from RPMI to DMEM. Do you have any papers about this? Maybe I can show it to my supervisor. Thanks!

i am also wondering, is there a reason of why you cannot switch media just like that ? My cells are maintained in DMEM and then after seeding I keep them in MEM for 3 weeks (because my predecessor had an assay that was working for MEM)with media changes every 2-3 days. MEM is essentially similar in composition to DMEM except that DMEM contains approximately four times more nutrients than MEM, so I have been doing that for a while, and things have been responding fairly alright. But there are other characters of the cells that does not seem to be as great as I wish them to be, but the problem seems to be consistent and no matter what media I kept them in, it doesn't seem to be working.. so I am wondering if it is because I switch the media?

It is entirely possible. Essentially when you have cells in culture, you have adapted them to certain conditions, changing those conditions drastically may result in odd behaviours from the cells.

paper: here

While you are doing the change of medium, you should observe your cells to ensure that they have not changed their morphology and growth rates (and if you are interested in them - expression of your target genes/proteins and controls).

Hi Bob1,

you brought up an interesting point. I have always subcultured and maintained the cells in DMEM, and when I seed them, I differentiate my cells for 3 weeks in MEM. What I noticed though as the passage number increases, the cells appear to grow faster, be it the cells subcultured in DMEM or the ones seeded and differentiated in MEM. Morphology wise they look the same under the microscope. Come to think of it, the cells was in MEM when we bought it, and we added it straight to DMEM, so it's possible that the cells are still adapting to the new media thus the increased growth rate.