MC3T3-E1 osteogenic differentiation
Posted 28 March 2009 - 11:02 AM
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!
Posted 31 August 2009 - 12:21 AM
Edited by zodiac1505, 31 August 2009 - 12:23 AM.