MC3T3-E1 osteogenic differentiation - (Mar/28/2009 )

Hi,

I'm currently trying to differentiate MC3T3-E1 pre-osteoblasts. It seems like a pretty common protocol, but I haven't been successful in getting mineralized tissue (assessed by von Kossa staining).

My current protocol is:
- seeding density: 250,000 cells/well in a 6-well plate (so about 25,000 cells/cm2)
- differentiation medium: alpha-MEM supplemented with 10% FBS, 100 U/ml penicillin, 100 ug/ml streptomycin, 50 ug/ml ascorbic acid, and 10 mM beta-glycerophosphate
- The osteogenic supplements are added fresh during each medium change, and I think they're prepared correctly because when used in the osteogenic differentiation of primary rat bone marrow cells, they work well.

I understand it takes about 14-21 days for mineralized tissue to form. However, currently at day 11, I'm still not seeing any morphological differences between the induced and non-induced cultures. Are there supposed to be morphological differences by this time? Also, does anyone know how does mineralized tissue from MC3T3-E1 cells compare with that from primary rat bone marrow cells?

Any advice would be greatly appreciated!

I did some differentiation long time back. I used both MC3T3 and 72F cell lines. The culture media and the technique was the same as you are using though we used less number of cells per well. With both cell lines, I used to do alkaline phosphatase staining (early indicator of differentiation) followed by von Kossa staining. I was able to see alkaline phosphatase staining starting from Day 15 for MC3T3 cells but couldn't see any von Kossa staining even on Day 24. But with 72F cell line, there was strong staining for both alkaline phosphatase (starting Day 3) and von Kossa (Day 10) and we could see black spots on the plates with the naked eye even before staining. I have no idea why the MC3T3 cells failed to stain for von Kossa. Maybe you could try with 72F cell line as they differentiate much faster compared to MC3T3.