Development Of Continuous Density Gradient Cell Separ.

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A compact bench-top model of the centrifuge enables continuous cell separation according the density. The apparatus holds a small separation disk equipped with a circular channel (8 ml capacity) interrupted by a septum. A set of isotonic Percoll media with different densities was continuously introduced at one terminal and collected from the other. Under centrifugal force field, cell suspension introduced into the proximal portion of the channel results in continuous separation of cells according to their densities. The performance of the apparatus was demonstrated on separation of human buffy coat containing nucleated cells of more than 100,000,000 among a large population (10,000,000,000) of RBC. The differential leukocyte count on a smear preparation with May Grunwald-Giemsa stain revealed that basophils were concentrated as high as 15% in a fraction at density = 1.075 g/ml. The flow cytometric analysis showed that CD34 positive cells were mainly collected in a fraction (density = 1.060) mixed with monocytes and lymphocytes. The method has been successfully applied for separation of cultured cells (over 1,000,000 cells) where human mast cells (density = 1.085) were well separated from large fibroblasts (density = 1.065) with minimum cell damage. The present method may be efficiently applied for enrichment of hematopoietic stem cells from cord blood.