we are trying to differentiate MG-63 pre osteoblasts into osteocytes using the differentiaing media (containing Dexamethasone, . Sodium L - ascorbate and Beta-Glycerophosphate disodium salt hydrate) but the cells are detaching as they continuously proliferate. is their any solution for the same?
we are doing it in 12 well plate, seeding 60,000 cells in each well.
we are letting the cells grow in normal MEM media till 70% confluency, then replacing the media with the above mentioned differentiating media, and changing the media on every 2nd day. cells are suppose to stop proliferation and start proliferation when switched to differentiating media.
These are not preosteoblasts, but a poorly charecterized osteosarcoma line that has been propagated in culture for many years. I cannot see how they could ever be used for such a purpose.
MG63 cells are osteosarcoma cells, being essencially a proliferative cell population. They do not folllow the normal proliferation/differentiation osteoblastic pathway, namely the reciprocal but inverse relationship between proliferation and differentiation. Certainly, they do not differentiate into osteocytes.
I have worked with this cell type and they are not valid for any differentiation experiments in general.
Interestingly, it is possible induce ALP activity in MG63 when supplementing with vitD3 and TGFb1 (Bonewald L). According to some publications from late 80's it was possible to select a subclone MG63.3A which showed increased adhesion, slower proliferation and ability to mineralise matrix in vitro (Dedhar S). However, there was not much work on this later on...
MG63s won't differentiate into osteocytes.... the closest to an osteocyte derived from a cell line is Linda Bonewald's MLOY4 line which has been used by the guys below (in conjunction with MG63s to create an interesting osteocyte model in vitro
Front Endocrinol (Lausanne). 2014 Dec 9;5:208. doi: 10.3389/fendo.2014.00208. eCollection 2014. A new method to investigate how mechanical loading of osteocytes controls osteoblasts. Vazquez M1, Evans BA2, Riccardi D3, Evans SL4, Ralphs JR3, Dillingham CM5, Mason DJ3.
MSCs will easily differentiate into osteoblasts with several variations of the differentiation media that you have proposed and are easy cells to work with. To become osteocytes, osteoblasts must first become embedded in their organized mineralized ECM and then go through a further differentiation pathway to become osteocytes. The exact nature of this pathway is not fully understood, but the following article addresses some of the current thoughts on it.
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2981593/
Maybe you can give us an idea?
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