How to convince suspension cells to adhere more tightly?

Question

I'm developing a cell-based assay in 96-well plates that requires adherent cells, as they need to be washed at least twice during the protocol. I'm using in-house strains of [HT1080](https://www.atcc.org/Products/All/CCL-121.aspx) cells (some overexpressing a certain protein of interest) that unfortunately have been selected for suspension growth in chemically-defined media (50/50 [CD-CHO](http://products.invitrogen.com/ivgn/product/10743011?ICID=search-product)/[CD-293](https://products.invitrogen.com/ivgn/product/11913019?ICID=search-product)). I can convince them to adhere by culturing in [DMEM](http://www.invitrogen.com/site/us/en/home/Products-and-Services/Applications/Cell-Culture/Mammalian-Cell-Culture/Classical_Media/dmem.html) with 10% [FBS](http://products.invitrogen.com/ivgn/product/16000036?ICID=search-product) and growing them in [CELLBind](http://www.corning.com/lifesciences/us_canada/en/technical_resources/surfaces/culture/corning_cellbind_polystyrene.aspx) flasks, but they still tend to pull away if there is very much shearing force, such as during the wash steps, or when they're in PBS during the assay wash and incubation steps (maybe 2 hours total for now, I'm still optimizing that).Unfortunately, the parameters of my experiments don't allow me to keep the cells in serum-containing media during the assay, as it would interfere with the uptake of my drug. Like I've said, when in PBS they start detaching, and I would assume the same in HBSS, which I don't really want to use anyway due to its low buffering capacity in CO₂ incubators. Might DMEM alone (without FBS) provide a little more impetus to stay attached during the assay? What would the effect be of increasing the amount of FBS to say 15 or 20% during culturing - would that promote stronger attachment? I'm a little hesitant to coat the wells with poly-*D*-lysine, as my readout is on a fluorescent imager, and I'm worried it would substantially increase background. I don't really have enough time to re-select the cells for adherent growth (although if I'd known about these issues from my predecessor 3 or 4 months ago I would have started right away...), so are there any other tricks I can try in the meantime? Are there any media supplements I could use to promote adherin expression or something like that? Any tips or advice at all would be appreciated.

Answer 1

This is just a suggestion, as I have worked with tissue culture cells in the past, but never without FBS to allow the cells to adhere to the surface of the assay plate. Can you design an experiment that tests the concentration of FBS in varying amounts from 5% up to say 15%. If you are culturing them in FBS and using DMEM can you try doing your assay in DMEM if PBS is the issue? Also, I assume when you are changing the media that you are replacing the new media by not directly pipetting onto the surface of the plate, but rather the side.

This sounds like a trouble shooting issue unfortunately. However, I would start by designing a dose concentration FBS experiment. I am wondering if there is a small dose that you can use that allows the cells to adhere and not inhibit the uptake of the drug. Best of luck to you, I know how much it can be a struggle and such a time consuming endeavor tissue culture work is!

Answer 2

Try filter plates instead of encouraging attachment. You will change the gene expression if you adapt non-adherent cells to attachment.