Cell Separation Techniques

Silvano Ferrini and Lorenzo Moretta, Istituto Nazionale per la ├▒icerca sul Cancro, Genoa, Italy

The past three decades have witnessed major progress in the development of methods allowing the identification and separation of various cell populations. This has had a major impact upon the precise dissection of the immune system and the definition of the cellular basis of the immune response. The combined use of newly identified cell surface markers and cell separation techniques has been fundamental for the precise assignment of given functional properties to well-defined cell subsets.

The first cell separation techniques, which were developed in the early 1960s, were based on physical differences such as cell size and density. The limitation of these methods is mostly related to the low degree of physical heterogeneity existing among lymphocytes. However, this approach may be parti cularly useful when combined with techniques which make use of phenotypic markers to tag the cells so that they can be separated by physical means (e.g. rosetting techniques). Major advances in cell separation techniques were made possible by the identification of membrane receptors or surface antigens which are differently expressed by lymphocyte subsets. For example, the identification of immunoglobulin (Ig) molecules on the surface of B lymphocytes and of receptors for sheep erythrocytes on human T cells allowed the identification and the separation of B and T lymphocytes, respectively. More recently, the development of monoclonal antibody (mAb) technology has provided a potent tool to precisely and reproducibly identify lymphocyte subsets expressing given antigens. Most of the mAb-defined surface leukocyte antigens have been classified into a still growing list of 'cluster of differentiation' (CD) antigens. Therefore, at present, the most widely applied lymphocyte separation techniques are represented by immunoselection procedures based on the use of mAbs. Among immunoselection techniques, the use of mAbs and complement (immunotoxicity) allows the selective depletion ('negative selection') of cells expressing a given surface antigen. Other immunoselection techniques, however, allow both 'negative' and 'positive' selection. Most of these techniques are based on the attachment of antibodies to different substrates or carriers, including plastic surfaces, erythrocytes or magnetic beads. Cells that are recognized by these mAbs and thus bind to these substrates can be subsequently separated by adherence, density gradients or magnetic fields, respectively. There is little doubt that the most precise and objective tool for the separation of cells stained with fluorochrome-labeled mAbs is represented by the fluorescence-activated cell sorter (FACS). A major advantage of FACS fractionation over the other immunoselection procedures is represented by the possibility of precisely defining the density of a given surface antigen and, therefore, the threshold values for cell separation. In addition, the FACS allows simultaneously analysis of several parameters and the separation of cells accordingly. In spite of these major advantages, a limitation of the FACS is related to the relatively low numbers of cells that can be processed per unit of time.

When approaching a problem of lymphoid cell separation, the choice of the technique to be applied should take into account a number of parameters, including cell numbers, purity and yield. In most instances, cell purity is inversely related to the yield. Less purity should also be expected when the cells of interest represent a minor fraction of the initial population. In addition, the possible consequences of cell manipulations on sterility, cell viability and function should be carefully considered. In some instances it may be convenient to combine two methods, for example a rosetting technique (which allows the processing of large numbers of cells) followed by FACS sorting (to obtain a high degree of purity).

The principal methods of lymphoid cell separation are listed in Table 1 and are described below.

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