mouse bone marrow MSC culture - (Jul/26/2011 )

i am culturing the bone marrow cells of balb/c mice of 6-8 weeks. after 3 days of plating mesenchymal cells start appear(fibroblast like). But the MSCs do not multiply and after 14-15 days of culture, those cells die. I use DMEM+L-glutamine media. Please suggest me about how to get a confluent culture.

mouse BM MSC sometimes takes long time for them to start growing after isolation, so it sounds normal for me the isolated cells are not growing right after the isolation.
But in your case, cell is dying, so first of all, I will make sure the initial seeding density, then will check the medium (especially quality of FBS)

i am using DMEM low glucose 1x (gibco) and add Hyclone fetal bovine serum - 20%.

I flush bone marrow of 1 femur bone and seed in one 100mm dish.

please suggest what changes i need to make for cells to grow?

you only use one femur from mouse and seeding in 100mm dish?
I think the seeding density might be too low.

Do you normally count viable cells before seeding?

Medium sounds fine to me, but also have you ever seen any contamination?

Dear all, I could finally manage to grow the MSCs from bone marrow primary culture .

But, i face a new problem.
m unable to detach all cells from a T25 flask by Trypsin EDTA. I keep the trypsin at 37degree b4 using. I wash the cells twice with TPVG and add 2ml more TPVG and keep for 2-3 min at 37 degree but still all cells don't detach.
Due to repeated TPVG exposure cells are also getting stressed.


Please pls.. help.

thanks.

blossoms123.

Have you already tried giving the sides of the flask some slaps with your palm after trypsin exposure ? This mechanical influence can help (at least it did for me)

You could try incubating with trypsin longer. I've had trypsin go bad on me before as well.