Dear all,
I use Saos-2 osteoblast-like cancer cells on my research to assess the osteoconductive and osteoinductive properties of some biomaterials. Saos-2 cells divide great and their viability is fine, but whenever I start feeding them with the mineralization activation coctail (beta-glycerophosphate, ascorbic acid, and dexamethasone) about only 10% of the cells survive after a day; and, these remaining cells die after couple of days. I just try to get viable cells for 15 days, but I'm only able to make them live for up to 10 days.
All ingredients I use are cell-culture grade and as I said Saos-2 cells I have can grow and divide exponentially unless I add the above compounds in them. Cell death is not dependant on any of the biomaterials I use, they die on common polystyrene plates, too.
What do you think is the problem?
Hi
The problem that you have mentioned is really serious. How much time you are exposing cells to trypsin for detachment. Try to minimize the time of exposure and after that try to was cells at least two times for the residual removal of trypsin. The concentration of beta-glycerophosphate, ascorbic acid, and dexamethasone whether you have optimized ? If not then try to analyze this with toxicity assays. And check any chances of contamination too.
Good luck
Are your supplements sterile/filtered? Try doing the trials with the same supplements on other cells, if you have any suitable cell line (MC3T3, mesenchymal stem cells), to see whether it is a general cell beahviour or sth wrong's going with your supplements.
Dear Mamatha and Joanna, thanks for your comments. First of all, I cultivate these cells in DMEM with 10% FBS and they are all high-quality Sigma products. I do filter the growth medium again with a 0.22 micron PES filter after the addition of glycerophosphate etc. Normally what people do is add at least 5 mM of glycerophosphate, 50 ug/mL of ascorbic acid and 10 nM of dexamethasone (dissolved in high grade ethanol). I add ethanol in 1 uL in every 10 mL of DMEM, so it's effect is probably very minimal. I do use these concentrations.
I did do a toxicity test before and dexamethasone and L-ascorbic acid do not affect cells. But beta-glycerophosphate addition into the medium reduce the number of cells. This glycerophosphate is cell-culture grade, too.
Unfortunatelly, I do not possess any other thype of cell like MC3T3, which would have similarly behaviour with SaOS-2. I have breast cancer and colon cancer cells. Though those cells are not as differentiated as Saos-2.
Problem might be with trypsin addition on cells, but still, as I said, this cell death occurs only when cells are supposed to differentiate into their osteoblastic stage.
Could this be a problem with the high passage number? Or filtering ?
Dear Bora, based on what you've said, all is fine with the supplements, how about the cell density? Do you think the cells feel happy?
Nornally, let's say if the confluency is around 50% cells grow exponentially and they look happy, but they detach the moment after I add the differentiation medium.
However when the confluency is more than 80%, not all cells detach and die. Some remain viable. Though the number of viable remaining cells is low (let's say it drops to 30% confluency). Here I can understand that, for differentiation, confluency could be a need? Am I correct?
Yes, I think it also may be an issue, suggest playing with different cell density or checking the literature on Saos-2 osteogenesis, maybe there is sth which you actually are not aware of. Also, check whether DMEM is a good choice for those cells when differentiating- this is what comes to my mind. Here's one paper you can look at:
https://www.ncbi.nlm.nih.gov/pubmed/24959407
- Badges
- Science topic