Application of a novel continuous-flow cell separation method for separation of cultured human mast cells

A novel, continuous cell separation method, based on density differences, is developed for application to transfusion medicine. Performance of a miniature separation column with ca. 8 mL capacity was examined on separation of human huffy coat (containing 10(7) nucleated cells and 10(10) erythrocytes/mL). Differential leukocyte counts revealed that lymphocytes were concentrated in two fractions of density 1.065 to 1.070. Neutrophils were located in another fraction (density 1.080), while some cells present in early eluted low density fractions were difficult to identify by microscope examination. Flow cytometry analysis revealed that CD34-positive cells, which are considered to be stem cells, were relatively concentrated in an early eluted fraction. Comparing the data obtained from hemocytometer and flow cytometry suggested the presence of immature CD45-negative nucleated cells in the same fraction. These results indicate that the present density method might be capable of collecting CD34-negative stem cells from peripheral blood. The present method was applied to the separation of cultured human mast cells. When the co-cultured cell suspension containing 3.5 x 10(6) cells (approximately 10% of mast cells and 90% of fibroblasts) was separated with a set of media (density: 1.065-1.085); fibroblasts were concentrated in a fraction of density 1.065 with a few young mast cells. Densely granulated matured mast cells were collected in a fraction of density 1.085. The method could also be used to detect minute changes in cell density under various physiological and pathological conditions.