I’m planning to seed canine chondrocytes onto coverslips inside a 12-well plate prior to immunocytochemistry in order to investigate the differentiation and collagen II production of these cells. Has anyone tried this before and has experience with a good coating material to attach the cells? We usually use collagen coated coverslips but I suggest this won’t work with chondrocytes because these cells change their phenotype and matrix production when grown on collagen.
Article Integrin Expression and Collagen Type II Implicated in Maint...
Collagen coated would probably be best as the cells are attached to collagen in vivo and plating on collagen does slow down the dedifferentiation process. Type II would theoretically be best but type I will probably be fine as its unlikely that the cells will recognise the subtle differences between I and II when plated in monolayer. Chondrocyte phenotype is mainly affected by being taken out of a 3D matrix and put into monolayer culture so any coating will be better than glass or plastic
First, thank you for your quick response. Our Problem is, that we don’t cultivate our cells on a 3D scaffold but in uncoated Well-Plates in monolayer culture. We now want to check their phenotype and therefore, we plan this IHC for typical chondrocyte marker molecules. I’m concerned that growing them on collagen coated plates would maybe distort the results. What do you think? Has anyone tried to grow them on glass or plastic without coating?
I have tried EZ-slides from Millipore. They are great and easy to use and then you can disassemble them and used them for immunocytochemistry staining. The problem of growing then in monolayers is that (as John mentioned) the phenotype will be affected. But maybe you can try growing them for few days only.
I have used 0.5% gelatin (Life Technologies) coated coverslips for primary cells (mouse calvaria) and cell line (HeLa cells). It worked very good. I would recommended to use this.
Thanks
Dear Nadja, It would be good to know the type of chondrocyte (primary vs. propagated, articular vs. growth plate, from juvenile vs adult articular cartilage) and culture media (serum vs. serum-free) you are planning to work with as that – at least from my experience - can have quite some impact on the cell’s in vitro behavior. It sounds as if you only want to replicate your experimental conditions in an IHC-compatible format. In this case I would not bother using any type of additional coating, which will introduce a new variable to your system. If your cells attached onto tissue culture-treated plastic (TCP) (or more precisely, they attached to serum proteins bound to TCP or glass etc.) they will do so on any tissue-treated cover slips. There are plenty of options out there. Nunc’s Thermanox worked for us. BTW, if your institute owns an inverted (fluorescence?) microscope I would simply do culture, staining and imaging in “normal” well plates. I normally use glass or TCP chamber slides for this purpose. The chamber slides can also be used on confocal or any other microscope if necessary. No matter what you do, though, keep in mind that primary articular chondrocytes upon attachment to any two dimensional surface inevitably dedifferentiate and adopt a proliferative fibroblastic phenotype. I believe maintaining or even promoting chondrogenic differentiation under such conditions is rather impossible, unless you seed them at a very high density in a micromass culture (e.g., ~200,000 cells in a 10 uL droplet into a well and left to attach for 2 h before addition of more media), which practically is a form of three-dimensional culture but probably much different from your original experimental conditions.
- Badges
- Science topic