A MICRO AGAR CULTURE SYSTEM FOR CLONING HUMAN ERYTHROPOIETIC PROGENITORS INVITRO

Abstract

A simple and reproducible micro agar culture method for cloning erythropoietic progenitor cells from human bone marrow is described. Mononuclear cells (MNC) were immobilized in an agar layer and stimulated by erythropoietin (Ep), which was added to a liquid overlayer. The cultures were routinely incubated, fixed, transferred to microscopic slides, dried and stained. Erythroid colonies were morphologically examined. The dynamics of growth observed from days 2 to 26 of incubation in the presence of 2.4 U Ep/ml showed basically 3 types of aggregates, which reached maximum growth on different days of incubation. A close Ep dose-response relationship was found for CFUE [erythroid colony-forming units] and BFUE [erythropoietic burst-forming cells] at a concentration of 7.5 .times. 104 seeded cells. By varying the plated cell concentration between 2.5 and 10 .times. 104 cells, a linear increase in the aggregates formed was found. On the basis of their growth dynamics and morphological composition, the existence of 3 populations of erythropoietic progenitor cells in human bone marrow is tentatively proposed.