added to the coated flasks at approximately 1 u00d7 10 8 cells t150 flask the cells were incubated in 15 mls of complete bone marrow medium at 33 u00b0 c in the presence of 5 co 2 after 3 u2013 4 days or when the stromal cells have adhered to the inner surface of the tissue culture vessel 15 ml of complete medium was added to the cultures dropwise so the cells were not disturbed one week later before vital components within the medium are depleted the so called u201c conditioned medium u201d i e the medium in the flask which contains non adherent cells was removed and 15 ml of fresh complete medium was added to the flask the non adherent cells were pelleted by centrifugation at 500 u00d7 g for 5 minutes resuspended in 15 ml of conditioned medium and returned to the original flask thus the non adherent cells were returned to the flask and the medium was changed in such a way that it contained one part fresh medium and one part conditioned medium in general the key to this regimen of cell culture is to 1 coat the inner surface of the tissue culture vessel with a solution of gelatin 2 keep returning the non adherent cells to the culture when exchanging the medium 3 add medium that contains sufficient nutrients to sustain growth without removing all of the substances secreted by the bone marrow cells which enhance their growth 4 supplement the tissue culture medium with.