A simple method for enrichment of polychromatic erythroblasts from rat bone marrow, and their proliferation and...

To evaluate the effects of a variety of chemical, biological and physiological stimuli on erythropoiesis, in vitro assays using erythroid progenitor cells from humans or laboratory animals are well-known methods. On the other hand, little has been reported on in vitro assays using mature erythroblasts such as polychromatic erythroblasts. In the present study, we established a convenient method for enrichment of polychromatic erythroblasts from rat bone marrow and confirmed their development in vitro. To establish a method for the enrichment of polychromatic erythroblasts, bone marrow cells from 3- and 10-week-old rats were separated by discontinuous density gradient centrifugation using Percoll. As a result, polychromatic erythroblasts were most highly enriched in the bone marrow fraction from 3-week old rats at the density interface between 1.040 and 1.058 g/ml. The enriched polychromatic erythroblasts were then cultured in growth medium supplemented with 20% fetal bovine serum in the presence or absence of erythropoietin for 48 hr. During the culture period, cell proliferation and maturation to orthochromatic erythroblasts were observed, and intracellular heme contents were also increased. In particular, the culture in the presence of erythropoietin revealed higher proliferation of erythroid cells, and therefore might be more appropriate for in vitro experiments on the effects of various stimuli on late-stage erythropoiesis.