I'm trying to expand rat BM MSCs, using DMEM, low glucose, 10 % FBS (Hyclone) + glutamax, but I'm facing some detachment of cells specially after passage #6. Any advice for the best conditions?
Masi, thanks for your answer, but i would like to ask you, for how many passages are you using 20% FBS before switching to 10%?
Just to be clear: the initial plating of the cells into culture dishes after harvesting is P0 (this is in 20% FBS). After the cells reach 80-90% confluency the cells are split into larger flasks for expansion (still in 20% FBS) and become P1 cells. When the cells reach 80-90% confluency the cells are split and frozen down in 80% FBS, 10% DMEM/F12 and 10% DMSO and stored in liquid nitrogen. These are now passage 2 cells. When these cells are removed from liquid nitrogen and placed into culture they are P2 cells and are grown in 10% FBS. Hope this answers your question.
Good Luck!
Hi Dr/ Masi
this is very very clear :-), i will try this protocol in my next isolation...
thanks a lot for your quick reply
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