Development Of Continuous Density Gradient Cell Separation Method

National Heart, Lung, And Blood Institute

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Abstract

Cell separation and purification are the most important first step in the cell research. Although fluorescence activated cell sorting (FACS) is a popular method to purify functional cells, it requires a preparative separation of cell sources from tissues for the effective purification. We have developed a novel flow-through cell separation method in which five density gradient media provide a density gradient along a circular channel in a rotating separation disk. This system continuously separates a large number of cells into five fractions according to their densities. The method uses a centrifugal bowl equipped with a single circular channel, which is interrupted at one portion so that the liquid introduced from one terminal is quantitatively collected from the other terminal. Each terminal has six inlets or outlets. A set of Percoll density media with different densities is continuously introduced through inlets 2 to 6 and collected through the respective outlets at the same flow rates. The sample cell suspension is continuously fed through the inlet 1 and collected from the outlets 1. The centrifugal force and flow rates are adjusted so that the cells are distributed in the corresponding density layers before reaching the outlet of the channel and collected into 6 fractions according to their densities. In the present study, we demonstrated an efficient separation of basophils which are present in less than 2% in white cell population in the peripheral blood. The preparative separation by the conventional batch density gradient method is difficult yielding in no more than 10% purity among nucleated cell population. In order to demonstrate the capability of our method, about 10 ml of anti-coagulated peripheral blood was separated by our apparatus. Lymphocytes were enriched in fraction 2 (1.070g/ml) at about 96%, basophils were in fraction 4 (1.080g/ml) at about 63%, and neutrophils were in fraction 6 (1.090g/ml) at about 92%. Harvesting and washing of cell fractions were finished within 3 hours in a single run. This simple preparative cell separation method would facilitate the diagnosis of allergic diseases of the patient as well as the investigation of the physiological role of basophiles. Future applications may include: 1. Detection of cancer cells in the peripheral blood 2. Isolation of primitive cells from peripheral blood and bone marrow 3. Separation of pancreatic beta cells 4. Separation of various other types of cells and intracellular particulates. Reference: Hiroyuki Shiono, Shintaro Ogawa, Satoru Niwata, Tadashi Okada, Yoichiro Ito, Preparative separation of basophils, major effector cells in allergy, by a flow-through density gradient cell separation method, presented in the 6th International Symposium on Countercurrent Chromatography held in Lyon, France on July 28-30, 2010.