Hiroyuki Shiono

Application of a Novel Continuous‐Flow Cell Separation Method for Separation of Cultured Human Mast Cells

Abstract 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 buffy coat (containing 107 nucleated cells and 1010 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 3.5×106 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.

Single‐step enrichment of basophils from human peripheral blood by a novel method using a Percoll density gradient

We have developed a novel continuous flow-through cell separation method using a Percoll density gradient. This method can continuously separate a large number of cells into five fractions according to their densities. To apply this method to the separation of basophils, Percoll density gradients were modified to improve basophil enrichment. When a set of Percoll density gradients was prepared (1.071, 1.075, 1.080, 1.084, and 1.090 g/mL) the basophils in a healthy volunteer were enriched by an average of 23.1 and 63.5% at Percoll densities of 1.075 (fraction 3) and 1.080 g/mL (fraction 4), respectively. On average, the yield of basophils was 1.66 × 10(5) cells in fraction 3 and 1.61 × 10(5) cells in fraction 4 from 9 mL of peripheral blood. The expression of CD203c (cluster of differentiation 203c) on separated basophils was upregulated by anti-immunoglobulin E stimulation similar to basophils in whole blood. Histamine release induced by calcium ionophore was also observed in the separated basophils. The present method will be useful for basophil enrichment since it preserves their function without using counterflow elutriation and immunological reagents, and this method will be effective as a preparative separation for cell purification by flow cytometry.