Isolation and Short-term Culture of Mouse Splenic Erythroblastic Islands

We isolated and cultured erythroblastic islands (EI) from the spleens of phlebotomized mice using a combination of collagenase digestion, unit gravity sedimentation, and Percoll density gradients separation. The isolated EI were composed of surrounding erythroid cells and central stromal macrophages (M phi), which were identified by Forssman antigen. While 60% of the erythroblasts incorporated bromodeoxyuridine, the M phi did not. EI could be maintained on a plastic dish for a short period in the presence of erythropoietin. Two hours later, the central M phi spread well and bound to erythroblasts via cytoplasmic processes. One day later, erythropoietic activity on the M phi surface continued, although their processes had retracted. Some EI showed synchronized expansion of erythroblasts and others showed differentiation to reticulocytes. Two days later, about 50% of the EI still showed erythropoietic activity and most erythroblasts differentiated to the orthochromatic stage. On the other hand, the M phi secreted colony-stimulating activity during the culture. It was infrequently observed that erythroid and myeloid populations simultaneously expanded on a central M phi. These results indicate that this EI culture system is useful for studying interactions between the stomal M phi and hematopoietic cells.