I'm trying to isolate mouse bone marrow mesenchymal stem cells.
My simple version protocol :
1. Whole bone marrow flushing with syringe and PBS.
2. large tissues removed with cell strainer(70um)
3. remove RBCs using Ficoll solution. or RBC lysis with buffer
4. cells seeded on 150pi dish with 20ml of growth medium. (about 10^8 cells/dish)
5. after 4hrs, remove floating cells with pbs.
6. overnight incubation and wash floating cells after incubation.
7. culture cells ->> problem occurred. all cells die after some days.
I can see some attached cells and slightly attached cells(round shape but not detached after washing step) after washing floating cells
But these cells all die after several days.
(I tried using bFGF(10ng~20ng/ml), FBS 10~20%, no antibiotics, DMEM -> Alpha mem, add glutamine, using gelatin coating etc. )
after days, Cells have degraded membrane and I can see debris of cells
Can anyone provide some tips for culture BMSCs?
After ficol hypaque separation take the middle buffy coat give it a wash then plate it with complete growth medium (DMEM+15% FBS+1% Antibiotic). u should remove the peripheral blood cells after 6 hr. if u observed under microscope. otherwise don't do. continue like other cell culture. this will work
I would skip the RBC removal this step may potentially damage the cells and it is not necessary for the RBC would not to grow, and would be washed away during culture maintenance, on top of that if you do bone marrow collection properly you usually do not get too many of RBC anyway. Otherwise, bone marrow cultures grow well on DMEM or DMEM/F12, serum 20% is way too high but 5% worked just fine for me.
Hi
You could extend step 5 to 24 hours to allow cells to attached prior to washing RBCs.. allow the wash to be gentle