Osteogenic Induction of MSCs, keep peeling off - (Jun/10/2011 )

Hi,

I seed my MSCs at 1100 cells/cm2 in 24 well plate formats and induce osteogenesis (medium formulation: 0.1nM Dexamethasone, 50nM Ascorbic Acid, 10mM Beta Glycerophosphate, high glucose DMEM, 10%FBS, 1% PenStrep) and change medium twice a week, for 3 and 4 weeks, but find that most times I try this, the cells begin to peel off 2 1/2 weeks into induction. By the time the 4week time point comes along, the cells are 90% gone, and I can't do nice alizarin red stainings at all. Anybody have any idea why this is happening to my cells?

Thanks!

Sorry, i have no answer for your question. But i have a similar problem with my freshly isolated passage 1 or 2 mesenchymal stem cells. I seed 50.000 cells/6-well-plate and 2 days after seeding i feed them with freshly prepared osteogenic differentiation medium (50uM Ascorbic acid, 100 nM dexamethasone, 10 mM b-glycerophosphate). After 1 day of incubation when i observe the cells, i see that most of them died and others have a disrupted morphology. This does not happen when i use a differentiation medium of 1-week-old or so. Do you think this can be because percentage of one of the ingredients is toxic to cells. Or can it be because i dissolve dexa in absolute ethanol and this is toxic to cells when freshly prepared (i then dilute in with DMEM and then add to differentiation medium of course). Any idea?