A separation chamber to sort cells and cell organelles by weak physical forces. V. A sector-shaped chamber and its application to the separation of peripheral blood cells

A separation chamber of 90 ml effective volume is described, that incorporates a sector shape (to prevent wall sedimentation) and anti-vortex lamellae (to prevent swirling upon acceleration and deceleration) as well as a flow-diverter for undisturbed layering and fractionation. The chamber is run an ordinary table centrifuge and possesses a resolving power for equilibrium density centrifugation that seems unsurpassed. Peripheral human blood cells were separated in a Percoll gradient yielding in 1 single run the concurrent isolation of 99% pure monocytes, 99% pure lymphocytes and 99% pure granulocytes. Recoveries varied from 94% to 102%. By modifying the shape of the density gradient, either 99% of all monocytes collected were more than 92.5% pure and at the same time separation of small versus large lymphocytes was obtained, or substantial purification of neutrophils and eosinophils was achieved. The potential for simultaneous separation of monocytes, B, T cells and granulocytes was established. Good separations were obtained with buffy coats from 1 U of blood. The chamber also functioned well in the standard Böyum method for mononuclear cell preparation. In addition, a method for the rapid removal of erythrocytes (within 9 min) at unit gravity is described with nearly quantitative yield of white cells.