Cell Separation by Centrifugai Elutriation

-There is an ongoing need to isolate and purify ceIls in bivalve mollusc disease research. Many separation techniques differentiate cells solely by a single factor and usually require additives in the medium that may damage live cells. Centrifugai elutriation is a technique that separates particles by size and density simultaneously and can be conducted in physiological media without additives. Living cells can be separated without chemical damage and, because the procedure can be conducted aseptically, cells can be maintained in vitro after elutriation. A description of the technique, methods to determine flow rates and rotor speeds, and a preliminary separation of oyster hyalinocytes is presented. In molluscan pathology and immunology, meth­ cording to their rates of sedimentation. Separation ods to isolate and purify cells are needed, he they is proportional to the size of the ceUs and to the hemocytes involved in the immunity processes, difIerence between the densities of the ceUs and cells susceptible to infection by parasites, or the the medium. Thus, velocity sedimentation is par­ parasites themselves. These methods must be ticularly useful in separating ceUs of the same simultaneously quantitative and qualitative and density (pretlow and Pretlow 1982). In centrifugai must preserve the structural and functional integ­ elutriation, ceUs are exposed to a centrifugai force ritYof the ceUs for in vitro studies. while suspended in a selected medium which 1 Classical methods of cell isolation are generally f10ws continuously in a centripetal direction based on differential and isopycnic centrifugations (Pret!ow and Pretlow 1979). The Beckman Instru­ in a density (pressure) gradient. The techniques are ments system designed for this technique is efficient but present sorne disadvantages related to shown schematically in Figure 1. the chemical nature of the products used (sucrose, In the elutriation chamber, each ceU tends to cesium chloride, PercoU, meglumine diatrizoate, migrate to a zone where its sedimentation rate is metrizamide) and the physicochemical changes they exactly balanced by the f10w rate of the f1uid. induce (e.g., in ionic force, osmotic pressure, or Because the geometry of the chamber produces a viscosity) at the concentrations used (Rickwood gradient of f10w rates from one end to the other, 1984). Moreover, these methods generally require cells with a wide range of sedimentation rates can several stages of centrifugation and collection. be held in suspension. By increasing the f10w rate An alternative method, counterstreaming centrif­ of the elutriating f1uid in steps, or by decreasing ugation, was originated by Lindahl (1948) and later the rotor speed, successive populations of homo­ developed by Beckman Instruments under the name geneous cell sizes can be washed from the cham­ of centrifugai elutriation (McEwen et al. 1968). This ber (Figure 2). The major theoretical aspects of procedure, which can eliminate the disadvantages elutriation were developed by Lindahl (1948), previously cited, has been used to prepare different McEwen et al. (1968), Pretlow et al. (1975) and vertebrate cells, e.g., whole blood and hemopoietic Sanderson et al. (1976). ceUs, cells from brain and liver, transformed and Elutriation Methodology tumor ceUs, ceUs from testis and ovary (Pretlow and Pretlow 1979), and also to purify protozoan para­ Meistrich (1983) defined and studied the various sites such as Plasmodium sp. (Russman et al. 1982) methodological factors of centrifugai elutriation. or Eimeria sp. (Stotish et al. 1977). This technique is Preparation of Single-Cell Suspension efficient because it is rapid and because a large quantity of ceUs can be purified at once. Sorne ceU systems, such as those of peripheral blood, are already in single-ceU suspension and Principles of Centrifugai Elutriation may require only treatment to avoid aggregation Centrifugai elutriation is a form of velocity (Mackler et al. 1977). However, ceUs in organs sedimentation in which ceUs are separated acand tissues have to be dissociated, either by