Cytotoxicity for chondrocyte monolayer culture - (May/17/2007 )

Hi, I am working on the drug induced chondrocyte cytotoxicity experiment. I cultured chondrocyte in multiwell plate for 24 hours, and then refed with media containing different drug concentration. The problem is after I refed the cell a lot of cell were dead even for the control (media without any drug). I don't know why. Is there anybody have these a kind of experience or have some suggestions? Thanks!

---

Sorry for my late response.
I don't really have the answer, but some questions and suggestions.
Did you use primary chondrocytes or a chondrocyte cell line? And were from? Because if you use primary chondrocytes at a high density, they don't attach to the well. And the chondrocytes that do attach, will de-differentiate towards fibroblasts.
Have you really checked how many chondrocytes were present after you refed? And if they were really dead (with some kind of staining)? And were they still round or dedifferentiated?

---

I have experience with Rabbit articular chondrocytes (primary's) which we used to induce iNOS and test compounds which were potential inhibitors. My advice would be to experiment with your cells at between 60-80% confluence. The cells should attach to normal TC treated plastic.....no problem. When they reach confluence they do tend to lift off the wells more readily i.e. when washing with PBS. For drugs that were insoluble in aqueous solution then I used DMSO at below 0.1% concentration. Above this the DMSO was itself cytotoxic.
Another important variable that people do not think about is the volume in the well. Most of my work has been done in 6-well plates....volume 3ml (9.6cm2 surface area). 24 well plate has a surface area of 2cm2... 0.63ml/well.
Be gentle with washing and refeeding. I use a pipette rather than a pastette on a vacuum line.

---

Don't you get de-defferentiation? I mean, as soon as my primary chondrocytes attach to TCP, they loose their round morphology and start to produce more collagen I and less and less type II.

---

The cells were just a model for proof of principle experiments. For our initial research we were interested in any animal cells that we could use to induce iNOS. iNOS was induced via stimulation with LPS alone. We were not interested in collagen our marker was iNOS. We went from this to human primary chondrocytes and iNOS, this was one of the first papers to show reproducible iNOS induction in human cells.
We then were testing compounds in the lab which were potential iNOS inhibitors. Therapeutically to use in the clinic for patients in sepsis.

---

Thank you, now I understand why!

---