mesenchymal stem cells

[rajlakshmi]

Dear friends,
I would like to know various protocols which are used for culturing mesenchymal stem cells from bone marrow.
Regards
Rajlakshmi.

[rajlakshmi]

Please help me out through e mail.

[amitays]

I just started working with human MSCs and I would appreciate any information or suggestions regarding the three following questions.

1) I am trying to induce osteogenesis by 10 nM dexamethasone, 10 mM β-glycerophosphate, 50μg/ml L-ascorbate 2-phosphate. The von koss and alizarin red
Staining are positive after about ten days. But the cells start to peel from the flask. How can I prevent that peeling? What cause that?

2)The bone marrow stem cells I am receiving for research are separated just by adhesion, not by Ficoll gradient or antibodies. Since, I have to make sure that I am working with MSCs. I will appreciate information of the efficiency of such a separation technique? Or if there is any suggestions how to separate efficiently MSC after they are cultured (one passage).

3) How many passages and in what ratio the MSCs are cultured?

[That_guy_over_there]

Greetings,

To avoid detachment, try lowering dex to 1 nM, or reduce serum a little bit (9% instead of 10, for instance), or change the serum batch.

There's no problem in isolating human bone marrow MSCs by adherence, density gradient is great to get rid of red blood cells and increase the chance of adherence.

Human MSCs are usually seeded at around 2EXP5 cells per cm2 (primary culture), and at around 4,000 cells per cm2 for subculture. They should keep their stem cell properties for some, say, 4 passages or so.

Cheers,

This guy over here

[amitays]

Thank you very much, I will try your suggestions