I’m trundling along here in my new lab, still trying to get everything up and running. On the tissue culture front, things have been fraught for some time, what with delays installing the carbon dioxide and nitrogen tanks, with learning how to live in harmony with the experimental prokaryotes sharing the same air space – all while finishing off a few key applications for funding. Finally, however, I thought I had the system working smoothly, and was ready to perform my first real experiment: infecting bladder epithelial cells with patient bacteria isolated from fresh urine.
This is an absolutely straightforward procedure. I’m simplifying it a little bit, but in essence, you seed your cells onto a coated glass surface (such as the chamber slide pictured above), perform the infection and, some time later, wash the cells and then fix and stain them to inspect what the bacteria have done to the cells and vice versa. It will be my bread-and-butter assay, the basis for testing the genetics and cell biology of infection and also trialling some of the interventions we hope to discover and perfect. So in that sense it is also a bottleneck: the basic experiment without which little else can progress.
So I was pretty miffed when it became apparent that Step One of this simple procedure – the step so basic that you wouldn’t normally even spare it a thought – was just not working. In short, I could not get the bloody cells to stick to the slide. Now this isn’t some ultra-finicking primary cell we’re talking about here: it’s ‘T24’, a full-out transformed tumor cell line that’s been growing for yonks, that’s so happy in culture that you have to split it multiple times per week to keep it under control. These are cells that are obviously quite keen to adhere when the notion suits them. They are the Sultans of Stickiness. They have gorgeous, frilly pseudopods that fan out as they probe their environment and inch their way across the plastic surface, forming and dissolving sticky adhesions with ease as they go. I could gaze at them for hours under the microscope, admiring their luscious lamellipodia.
But on glass, the game was completely off. Many cells can’t grow directly on glass, so when that failed, I tried coating with poly-D-lysine, fibronectin, collagen, serum, and attachment factor. All failed: the cells remained round, clinging together desperately in increasingly large clumps that wafted around the slide like seaweed on the ocean floor before dying a tragic death.
The real mystery, however, began when I contacted the lab in Cardiff that had kindly posted me the cells in the first place. Yes, T24 grew just fine on glass, I was told. No, no coating was required. Yes, they were perfectly happy, in fact, on the precise make of slides that I was using.
So, Dear Readers – I throw the question open. What now? Can anyone help solve the mystery? Or will my line of research suffer…a sticky end?
(Sorry.)
Are you using exactly the same media as the Cardiff lab for the T24s? Same concentrations of supplements?
Assuming the Cardiff lab willing/able to help, and possibly unlimited money (hahh) I would get them to give you – collect or courier/post – a flask of growing T24 (in case they went weird during freezing/defrosting), plus a bottle of media with serum which they are successfully using to seed onto glass, plus (why not) one of the chamber slides that they used.
Serum batch effects messed me around so much in my PhD. Sorry if these suggestions are too obvious :). Troubleshooting is fun when it’s not your own work on the line…
Have you tried acid etching? (quick and easy withthis protocol)
Dave, I was thinking of trying that, so thanks for the protocol. Much appreciated.
Everything is the same, Miranda, except I’m not sure it’s the same brand of serum. I’ve never known serum brands to affect simple adherence properties – to me this doesn’t seem likely. I know anything is possible, though. I won’t have enough money to do any more shipping until I get a grant…so if my last few options fail, I’m just going to source another bladder epithelial cell line (there are several). Life is too short and the choice of T24 was arbitrary.
Jenny: I know that the serum source _shouldn’t make a difference_ but I got various results in my old lab changing from brand X to brand “lab we collaborated with”. At the time, I had almost given up trying to understand what was “wrong with the cells we used….” so I took it when the cells started behaving like they were supopsed to. I guess it could’ve been something I didn’t think about, but at the time the serum was the obvious difference.
The acid etching/stripping was my other suggestion, apart from the “shipping their slides and a fresh culture”. I guess there isn’t enough money for you to take a trip and go visit the lab and then bring some cells with you on the train? I hear Wales (that’s where Cardiff is, right?) is beautiful in the spring/summer 🙂
I hope you get the assay to work. I totally understand the “its a bread and butter assay but it just need to work” comment
I agree with the above (personal experience with serum lots and adhesion properties of “ultra-finicking primary cells”), but don’t bother with the acid etching if you can get new cells.
Two other possibilities are: they are tumor cells. Perhaps you selected for a newly aneuploid one, that now has different properties? Could be checked if you had karyotyping routinely, but you don’t, and I think it’s fairly unlikely. However:
Second is, mycoplasma. This is highly likely.
Sigma sells a PCR test for them; otherwise if your cultures are crawling with them, you can use this cheaper alternative (which I’ve never tried, myself, but have heard about for years). There are pictures all over of what positive cultures look like.
The point being, that persistent low-level mycoplasma infection can lead, over time, to chromosomal instabilities.
Anyhow, since the best solution, given these are cell lines, for the above problems is to get a new culture and start over, you don’t really need to know the reason, but it’s so unsatisfying not to that you may want to examine these possibilities even so.
I know that by putting all these links that this will be held up in moderation, but presumably you’ll be looking at the queue shortly 🙂
I’ve taken a pretty high-power look at these cultures using DAPI and microscopy and saw no evidence of mycoplasma. I know this can be missed without PCR, though. However, I’m using a direct thaw of a frozen vial of cells that stuck to glass the week they were frozen down, and they only went through two passages here before the experiment, so I do sort of feel that the explanation may lie elsewhere. I am guessing it’s probably something really stupid, like a bad batch of chamber slides. The selection pressure for good adherence is one of the strongest evolutionary pressures my tissue culture cells will be under, because I wash the crap out of them when I trypsinize, and also rinse off the first trypsin, which will select against anything that doesn’t really stick hard.
I’m getting different chamber slides, and will also try a second cell line. If that doesn’t work I know it’s something more sinister. As much as I’d like to understand the problem, I can’t spend a lot of time troubleshooting. And anything that won’t perform in Gibco’s standard EU-Gold FBS is out the door – I’m on a budget here. 🙂
Yeah, I suspect the glass itself. Have you got other cells, or can you try the T24s on glass coverslips in plastic plates?
Maybe you have discovered some incredible new and unexpected phenomenon…
Alexander Fleming, eat my fungi.
For God’s sake, if you have found something, don’t do a Fleming and ignore it.
Another, possibly naive, suggestion – are you SURE those are T24’s you’ve got there? You refer to their luscious lammelipodia, but since the cells you’re seeing down the scope don’t look like that…
Contaminating cell line of some kind?
Cardiff shipped you the wrong thing by mistake?
Somebody (not you) simply yanked the wrong vial from the freezer (either at your end or theirs)?
Yeah yeah, I know. Failing that, I’m going with Heather. We spend a lot of time karyotyping tumour cells, not to mention ES and iPS cells, as a QC measure since the wretched things like to rearrange their chromosomes in culture.
Bugger. “Lamellipodia” of course.
Richard, they are growing beautifully on plastic and have the correct morphological phenotype. It is only when I split them off onto glass for an experiment that they fail to adhere. So it’s not as if they’ve undergone some sort of weird mutation where they suddenly can’t stick full stop. And their lamellipodia are indeed luscious – when it suits them.
Unlikely to be the wrong vial – it was labelled with a machine and everything. And it was my lab’s First Ever Vial. (You always remember your first…)
I should know pretty soon if it’s my glass surfaces – I’m closing the deal on a new cell line and I’ve got new slides en route. Plus all sorts of fun stuff from Sigma like laminin.
Hm… see, I told you my suggestions were naive. 😉
The correct thing to do with at least one subculture re-frozen down from the First Ever Vial is to ensure that you keep it safely stored away for at least the next 20 years, regardless of how useless it may ultimately become. Refuse to dispose of it, citing sentimental reasons. If any punchy future grad student calls you out on it, deflect the question and/or hide it somewhere else.
This is part of the Grand Academic Tradition, you know.
Ha! You found me out! I have left vials over liquid N2 all over the nation by now. And don’t mention those boxes at -80…
Also, just wondering, why can’t you grow the cells on those Permanox plastic slides instead of glass? Optical properties? Autofluorescence?
Will definitely use Permanox/Thermanox if I have to, though in the past I’ve not been so enamored of the performance in confocal.
Winty, I plan to enshrine my First Ever Vial along with the Funky Monkey cells.
Thermanox is awful for confocal (but brilliant for EM). Do these cells of yours not grow on any glass, or is it just your fancy chamber slides? have you tried standard coverslips? Do other cell lines grow happily on your chamber slides?
I don’t have any other cells to try yet, though I’m working on it.
I’ve used this brand of chamber slide for years, though, it has to be said, it’s a new box so I can’t say for sure it’s not a lot failure.
If you put the cells in your chamber slides, and leave them overnight, and if they don’t stick, can you take them out and put them on plastic to see if they then stick to that? What I’m driving at is are the cells becoming really poorly in your slides and starting to die, or are they growing quite happily just not sticking?