A MINIATURIZED AGAR CULTURE SYSTEM FOR CLONING HUMAN ERYTHROPOIETIC PROGENITOR CELLS

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  • 1 January 1984
    • journal article
    • research article
    • Vol. 12  (1) , 75-79
Abstract
The micro-agar-culture technique for cloning early and late erythropoietic progenitor cells (BFU-E and CFU-E) was further modified and miniaturized in order to study the optimal growth conditions with a minimal consumption of erythropoietin (EP). Using microtiter plates, the total incubation volume was lowered from 0.5-0.1 ml and reduced the necessary amount of EP and cells by a factor of 5. The method consists of a 50 .mu.l agar layer, in which the mononuclear cells are suspended, and a 50 .mu.l liquid overlayer containing bovine serum albumin (BSA), transferrin (TF) and EP. After a 7- or 14-day incubation, the whole agar layers were fixed, transferred to microscopic slides, dried, stained using the Pappenheim method, and permanently preserved. The influences of FCS [fetal calf serum], BSA and TF in the presence of EP were studied on the proliferation of human bone marrow CFU-E and BFU-E. The variation of FCS concentration showed an optimum at 10%. The addition of BSA in the presence of optimal concentrations of EP markedly increased the number of BFU-E, but not CFU-E. The threshold concentration of EP required for the initial burst formation couild be reduced by half in the presence of BSA. By the addition of TF, a further increase in the number of BFU-E was obtained.