Why won't you grow for me?

October 1, 2008 6:59 AM

Specific Cell culture question:

I'm currently trying to culture the Dempsey cell line (ATCC# CCL-28) and am having no luck. The cells are just not replicating. They're alive but they don't repicate at all. We've double and triple checked the conditions described on the ATCC page and according to what's out there we should be golden. We've also checked a few lab pages and the references and everything matches what we're doing. Has anyone worked with these cells? I feel like there's something extremely obvious that I'm missing.

Here is our exact culture conditions:
McCoy's 5a Media from Gibco (or whatever company Gibco is now) supplemented with L-glutamine (1.5 mM) and sodium bicarb (2.2g/L) and 20% FBS (heat inactivated - is this the problem?)
37 deg C and 5% CO2
Cultured in 10 cm plates (Sarstedt) - uncoated (are we supposed to coat them with something?)

The L-glutamine is frozen in one-shot tubes and our other lines grow just fine, including a couple MES lines, so I don't think it's bad glutamine. The bicarb is culture-grade powder and, again, the other lines growing are just happy as can be so I don't think it could be that. We have no tested for mycoplasma but a test kit is on the way, just in case. Checking via DAPI has revealed no contamination but we're gonna do the PCR-based just to be extra sure. We haven't had any mycoplasma problems before but we've just received three new lines so I think a test would be prudent even if we weren't having issues with this line.

Thanks for the help everyone and sorry for the length.

posted by LunaticFringe to science & nature (10 answers total)

I haven't worked with these particular cells, but I think your intuition about trying another serum is worth following. I assume you've changed the medium or centrifuged to remove the DMSO in the freezing medium, and followed all the typical cell culture protocols. Double check that the plates are tissue culture polystyrene; if so, and if the cells are attaching to the plates, then the surface is probably OK and protein coating is unnecessary (and it's not mentioned in the ATCC protocol anyway).
posted by Mapes at 8:45 AM on October 1, 2008

I haven't worked with these cells either but after reading the ATCC info sheet on them, it looks from the recommended subculture ratio of 1:2-1:3 that they are slow growers that like to be almost confluent to be happy, with media changes a couple times a week. Have they adhered to the plate and are they fairly densely plated?
posted by violette at 10:38 AM on October 1, 2008

The plates are polystyrene and the cells adhere, in fact they look quite healthy except for the not-growing bit. I've also been changing the media on the plates regularly so the DMSO is long gone. Thanks for the suggestions though!
posted by LunaticFringe at 10:40 AM on October 1, 2008

(Should learn to preview) They've never been split less than 1:2 and have only been confluent enough to split once in 4 weeks of culture. I've also rewoken them in that time but since they're not growing I'm afraid to keep doing this as our LN2 stocks are getting too low.
posted by LunaticFringe at 10:42 AM on October 1, 2008

They could just be reeaally slow growers but it's possible that they need an appropriate substrate in order to enter cell cycle.

If you can "borrow" tissue-culture treated (gas-plasma) plates, try trypsinizing your cells and transfer them to those plates. Alternatively, if you can get your hands on some polylysine, you can treat polystyrene plates with them, then transfer your cells into the treated plates.

I've seen some cells that can adhere to polystyrene but clump up and/or grow more slowly (whereas the majority of cells I've come into contact with just won't adhere and grow to untreated polystyrene).
posted by porpoise at 11:34 AM on October 1, 2008

Rather than adding in the glutamine and bicarbonate yourself, you could try ordering the pre-modified/supplemented media, just to troubleshoot?

Agreed that they could be slow growers and need to be plated more densely at first in a smaller dish, especially if currently they're all spread out when you first plate them in a big 10cm dish.

Just some ideas...
posted by NikitaNikita at 8:37 PM on October 1, 2008

I'd try buying some premade media, like Nikita suggested. (I do work at Invitrogen, so that is a caveat) When you split, are you using a cell dissociation buffer? We had some problems once with scraping/ banging the flask on the table, and dissociation buffer really helped viability and cell happiness.

And of course, if all else fails, you can cheat! Try to stimulate cell growth by adding some cytokines to the media - EGF sounds like a good one for you. I usually work with blood, so my weapon of choice is IL-2, but EGF at like 10 ng/mL might work.
posted by wuzandfuzz at 10:43 PM on October 1, 2008

PS: the 10ng/mL is a total guess. If you have more questions, feel free to shoot me a mail.
posted by wuzandfuzz at 10:46 PM on October 1, 2008

If you're going to try cheating, adding a little insulin couldn't hurt (we grow up hard-to-keep-alive mouse hippocampal neurons with ~25ng/mL insulin and adding B27 [it's an 'undefined' extracted substance from human blood]).
posted by porpoise at 11:03 PM on October 3, 2008

Followup: After trying media and serum changes, we finally contacted ATCC. This cell line senesces after 30 passages and they had sent up passage 28. So, that's why the cells wouldn't grow. They've since sent us a new vial at passage 8 but I haven't woken them up yet. I imagine they'll grow fine...
posted by LunaticFringe at 8:25 AM on November 2, 2008