I would like to establish a human bone marrow stroma in culture but I need to prepare the LTCM.
In our lab, human mesenchymal stromal cells from bone marrow are cultured as described (http://onlinelibrary.wiley.com/doi/10.1002/stem.1432/abstract).
Mononuclear cells are isolated from BM samples using Ficoll gradient and plated at 1.3e5/cm2 in DMEM-low glucose with 10% FCS. After 3-4 days, non-adherent cells are removed and medium is refreshed. The medium is refreshed every 3-4 days until 90% confluency is reached, after which we reseed at 4000 cells/cm2 and continue the 3-4 day medium refresh.
The FCS batch effect surely plays a role here, so check a few FCS batches to see which one works for you.
@martijin this protocol is for hBM-MSCs culture, but i'm looking for the normal bone marrow stroma culture which i should use the Long term culture media not the basal DMEM-LG medium.
It sounds to me that you want to do Dexter cultures with human bone marrow. 20 years ago, I worked with Dr. Christa Muller-Sieburg out in San Diego estabishing stromal cultures from human bone marrow samples. The culture medium was Iscove's modified Dulbecco's medium with 30% fetal calf serum - NOT heat inactivated. Be sure to add fresh 50 uM 2 mercaptoethanol. The medium as defined by Dexter included hydrocortisone, but Christa had determined that HC inhibited hematopoietic stem cells, so we omitted it.
As to input cells, although Ficoll is a pretty standard way to remove erythrocytes, you will lose a lot of the stromal cells and their precursors when you do that, which are often associated with the bone marrow spicules. We preferred whenever possible to use the marrow pre-Ficoll and lyse the red cells with Gey's hypotonic lysing solution.
You can also use ACK [ammonium chloride:potassium bicarbonate - 8.3 gm ammonium chloride/1.0 gm potassium bicarbonate/0.4 gm EDTA in 1 L distilled water]. We typically made a 10x ACK stock and diluted to 1x fresh each day.
We seeded the cells at 100,000 cells/mL in tissue culture flasks, and allowed the stromal to develop, feeding weekly by removing the supernatant, and feeding with fresh medium. The stroma will be heterogenous, with fibroblast-like cells, adipocytes and macrophages. You can passage the stroma; we released the cells with Trypsin:EDTA.
You can seed non-adherent bone marrow cells on top of the preformed stroma at low density [10,000 per mL] and you will get myelopoiesis.
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