Counterflow centrifugal elutriation

Counterflow centrifugal elutriation (CCE) is a negative-selection isolation technique that can be used to isolate large numbers of purified monocytes. This technique has been described for almost 50 years (66). However, it is only over the last 25 years that it has been increasingly employed, due to the venue of the Beckman centrifugal rotor, which has improved elution parameters (67). The principle behind the use of CCE for cell purification is based on differences between cell populations in sedimentation velocity in relation to cell volume.

Cell shape as well as cell density can play a role in the effectiveness of cell purification methods. However, the latter two aspects have a less significant impact on the effectiveness of cell separation by CCE than on techniques based on isopycnic separation. Cells loaded in the elutriation chamber are subjected to two opposite forces: the centrifugal force (rotor speed) and the centripetal force (the flow rate) (Figure 2), Providing that the centrifugal force is maintained (i.e. a constant rotor speed), the increase in the centripetal force obtained by the steady increments of the elutriation medium flow rate forces the cells to exit the chamber according to their volume. One major disadvantage of the commonly used elutriation system is that the percentage of cell loss becomes very high when the machine is loaded with less than 10s mononuclear cells. This means that more than 100 ml of human blood is required to perform a good monocyte separation (68). This fact poses a limitation in the use of CCE for separation of

Elutriation Blood

Elutriation Boundary

Elutriation

Figure 2 Separation chamber of the elutriation apparatus. A schematic representation of cell separation in the 5 ml elutriation chamber, indicating the opposite forces to which cells are subjected, is illustrated.

Figure 2 Separation chamber of the elutriation apparatus. A schematic representation of cell separation in the 5 ml elutriation chamber, indicating the opposite forces to which cells are subjected, is illustrated.

small numbers of cells. However, to date, CCE is the most suitable separation technique to isolate monocytes/macrophages in cell suspension with high yield and purity providing a good preservation of their functional properties. Recently, a semi-closed CCE system has been developed (using a new large capacity Beckman JE 5.0 rotor with one interchangeable 40 ml or 5 ml separation chamber), to purify macrophages from mononuclear cell concentrates of healthy donors and cancer patients (69). The high capacity of the rotor allows the harvest of very high numbers of monocytes in a single run and does not require a Ficoll density centrifugation step. Protocol 10 describes the purification of macrophages by CCE.

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  • claudia
    What are the technique used in cce system?
    8 years ago

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