Electron microscopic study of erythroblastic islands obtained by 'tissue-stamp culture' method

A new 'tissue-stamp culture' method was developed for stamping proliferating erythroblasts of mouse spleens on collagen-coated coverslips after inducing haemolytic anaemia by administration of 1-acetyl-2-phenylhydrazine, and then adherent splenic cells were cultured for a few days. We could obtain many erythroblastic islands, where cultured erythroblasts were located over macrophages and were proliferated synchronously for 10-30 h, and then the erythroblasts were differentiated and enucleated after 30-50 h in the presence of erythropoietin. To observe three-dimensional structures of the erythroblastic islands, a scanning electron microscope was used for the cultured cells treated with critical point-drying method. Immature wrinkled erythroblasts with many micropinocytic pits were attached to the central area of the flattened macrophages with many cytoplasmic projections, though matured erythroblasts were localized on their peripheral areas. Moreover, cytoplasmic projections of underlying macrophages, which were attached to the matured erythroblasts, were decreased in number. At a late stage, deep cytoplasmic invaginations of erythroblasts observed at a middle stage became shallow after their enucleation and flattened to form their concave shapes. This 'tissue-stamp culture' system would be useful for studying specific interaction between stromal macrophages and haematopoietic cells.