mouse bone marrow culture - mesenchymal stem cell - Cell culture (Feb/01/2005 )

Hi,

Anybody experience with mouse bone marrow culture?

I have isolated mouse bone marrow cells from tibia and femur of hind legs using DMEM low glucose or Iscove’s Modified eagles medium (IMDM) but so far I have not been able to maintain them in culture (incubator 37C and 5% CO2). I used the following culture/maintenance mediums:

DMEM low glucose + 20% FBS, 0.01% PS
IMDM + 20% FBS, 0.01% PS
DOM medium (IMDM + 15% heat inactivated FBS + 15% heat inactivated Horse Serum + 1uM hydrocortisone + 0.1mM beta-Mercaptoethanol + 200mM L-Glutamine + 0.5% PS)
alpha-MEM + 10% FBS + 1% L-glutamine, 1% PS

The mBM cells are either fresh or frozen in cryomedium (40% DL, 40% FBS and 20% DMSO). In the entire mediums above, there are a portion of the token cells which adhere to the flask surface (Falcon Primaria 25 cm2) while the other portion is floating cells. Some of those adherent cells have fibroblast morphology, some are compact round shape and others of strange morphology!!

I am seeking to expand the adherent mBM cells up to 5-8 passages. In the above culture medium, there are a few colony-forming-units that appear but the flask never reaches confluence. 30% confluence takes 3 weeks or more, for 1 million cells per ul culture medium seeded.

Help would me very much appreciated!!

hi there!

i know a guy around here who successfully isolates and cultivates bonemarrow-derived macrophages from mice. if you're interested, i can ask him what media an conditions he uses...

mike

British Journal of Haematology, 2003, 123, 702–711

Murine marrow-derived mesenchymal stem cell: isolation,
in vitro expansion, and characterization

Check this paper out; they use simpler a formulation for their primary medium (DMEM with 10% FBS). Cell seeding density of flushed marrow-flushed cells is 1.94E6/cm2.