Separation of cells by velocity sedimentation

TLDR

A sharply‑defined streaming limit restricts the maximum cell concentration in the starting band that allows useful velocity‑sedimentation separations. The study presents a system for fractionating living cells by velocity sedimentation in Earth’s gravity. Cells are loaded into a shallow 3–30 % fetal calf serum gradient at 4 °C, where separation occurs mainly by size (independent of shape) and the sedimentation velocity follows approximately r²/4, as verified by electronic counting and pulse‑height analysis. Streaming limits can be raised by optimizing gradient shape, as shown for sheep erythrocytes (limit 1.5 × 10⁷ cells ml⁻¹) where measured and calculated velocities agree, and the method fractionates mouse spleen cells by size with velocity ≈ r²/4, indicating potential for separating functionally distinct cell populations.

Abstract

Abstract A system for fractionating populations of living cells by velocity sedimentation in the earth's gravitational field is described. The cells start in a thin band near the top of a shallow gradient of 3% to 30% fetal calf serum in phosphate buffered saline at 4°C. Cell separation takes place primarily on the basis of size and is approximately independent of cell shape. A sharply‐defined upper limit, called the streaming limit, exists for the cell concentration in the starting band beyond which useful cell separations cannot be achieved. This limit, which varies with the type of cell being sedimented, can be significantly increased by proper choice of gradient shape. For sheep erythrocytes (sedimentation velocity of 1.6 mm/hour) it is 1.5 × 10 7 cells/ml. Measured and calculated sedimentation velocities for sheep erythrocytes are shown to be in agreement. The technique is applied to a suspension of mouse spleen cells and it is shown, using an electronic cell counter and pulse height analyzer, that cells are fractionated according to size across the gradient such that the sedimentation velocity (in mm/hour) approximately equals r 2 /4 where r is the cell radius in microns. Since cells of differing function also often differ in size, the system appears to have useful biological applications.