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LONG TERM HUMAN BONE MARROW CULTURE (LTHMC) USING BONE MARROW ASPIRATES FROM CHILDREN

To investigate whether small volumes of marrow aspirates are suitable for LTHMC, we cultured 22 marrow aspirates from children with a variety of hematological disorders according to the method of Coulombel et al (Blood 62:291, 1983). 5-40×106 buffy coat cells recovered from 3-4 ml of marrows were cultured in α medium with 10−6 Mol hydrocortisone, 12.5% horse serum and 12.5% fetal calf serum in 35mm dishes at 2×106 cells/dish. Demidepopulation of nonadherent cells was done weekly. Differentials on nonadherent cell showed early and late myeloid cells till 5th week, after which 95-98% of the cells were macrophages. An increase in the lymphocytes proportion for the first 3-4 weeks was observed. Studies of 3 specimens with monoclonal antibody OKT-3 revealed positive cells in 0 of the 3 nonadherent fractions, and 0.1% positive cells in adherent fraction in 1 at 2-3 week of culture. Adherent cell layer with nests of small round cells in "cobblestone-like" arrangement was established in 18 of 22 specimens within 2 weeks. There was clear association between the presence of the cobblestone-like cells and generation of CFU-C colonies by adherent cells. None of 5 adherent specimens without these round cells formed CFU-C colonies, while 3 of 3 adherent specimens with the cobblestone-like cells formed 5-9 colonies/5×104 cells plated. We conclude that establishment of LTHMC is possible with a small volume of aspirated marrows from pediatric patients, and that presence of cobblestone-like cells indicates generation of stem cells in the adherent layer.