A centrifugal elutriation system of separating small numbers of cells

Abstract

One of the major disadvantages of centrifugal elutriation (CE) is the relatively large volume (150 ml) of the various fractions, especially if small numbers of cells have to be separated and the fractions contain few cells. To reduce the volume of the fractions 2 elutriator rotors were coupled in series. Since the rotor speed of the second rotor was always kept 750 rpm higher than that of the first rotor, cells elutriated from rotor 1 were collected in rotor 2. After elutriation of a complete fraction from rotor 1, and collection in rotor 2, the cells were harvested from rotor 2. This was achieved by means of a flow distribution unit (FDU), which made it possible to disconnect the flow of both rotors and simultaneously reverse the flow of the second rotor. It is demonstrated that 40-95 X 10(6) mononuclear leukocytes may be fractionated without loss of resolution in fractions of only 9 ml. The lymphocyte (greater than 99%) and monocyte subpopulations (88-94%) obtained were as pure as with CE carried out with only 1 rotor. In addition, the cells in rotor 2 could be washed and suspended in culture medium prior to harvesting by means of the FDU. In this way loss of cells by additional centrifugation steps was avoided. Erythrocytes (RBC) present in certain lymphocyte fractions were lysed with NH4Cl and after lysis of the RBC and elution of ghosts and debris, the cells were washed and harvested. This procedure did not affect cell viability and the PHA response of the lymphocytes. The versatile system described made it possible to apply CE for the separation of small numbers of cells without loss of resolution, and demonstrated that CE is ideally suitable for concentration and washing of cells, and removal of contaminating RBC, not affecting the recovery, viability and function of the cells.