I have been growing a human foetal osteoblast cell line and primary bone marrow derived osteoblasts. During long term cultures (particularly mineralisation) these cells tend to fold over at the edges, creating clumps in the center of treatment wells. This affects the quality of any results gained and can even lead to the clumps loosening and being removed during feeds. I wondered if anyone had any tips for this? Specifically, does coating the treatment wells first help with long term attachment?
I have tried: lowering seeding density and FBS percentage (to limit growth), different methods of feeding to reduce cell disturbance.
Coating of wells: I was considering poly-l-lysine or collagen.
Many thanks in advance for any help,
Matt
Hey, Mathew.
I've had problems with getting cells to attach to glass. The solution is usually a combination of Poly-D-Lysine (I suspect Poly-L should work just as well) and an extra cellular matrix (ECM) protein.
Currently, I've been trying to capture images of Prostate Cancer Cells (PC3) undergoing Apoptosis. Poly-D-Lysine helped the cells attach, but the attachment was not strong enough such that the cells remained attached during apoptosis.
So then I tried coating the glass cover-slips with Fibronectin AFTER first coating it with Poly-D-Lysine. For the PC3 cells that worked. But in the past I've found that the exact combination of protein changes with the type of cells one is working with and the material to which the cells must attach.
I've not worked with osteoblasts before (although I'd very much like to), but "IF" I were to culture osteoblasts and ESPECIALLY the primary derived from marrow osteoblasts, I would try different combinations of the Poly-Lysine with:
1: Fibronectin
2: Elastin
3: Laminin
4: Collagen
AND if I still needed better adhesion than what was achieved, I'd add a CAM (Cell Adhesion Molecule) to the coating material. Osteoblasts express CAM receptors. I'm not an expert on osteoblats and I'd have to read up on them, but I believe I'd find a CAM that helped the osteoblast anchor in place without triggering a signalling cascade of some sort.
I'd be very interested in hearing how this works out for you. Would you mind contacting me and letting me how your results turn out?
Hey Tim,
What a fantastically helpful response! I think I will definitely order poly-l-lysine and couple its use with collagen or fibronectin (or some other combination), as you suggested. I suspect the marrow cultures will be easier to work with, as despite slower growth they almost always give more repeatable results than the cell line. I will definitely let you know how the results turn out, though it may be some time till I reply as the cells take so long to grow (not to mention time taken to place an order!).
Thanks again.
Try chondroitin sulphate as coatings, they attached and clumps could be avoided
Best.
Mannan
I would definitely prefer a coverslip coated with mixture of hyaluronic acid and collagen in the ratio of 5:4 for long term attachment.
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