Introduction

Cell-enrichment processes not using fluorescent or magnetic technology are based on cell physicochemical properties, such as density, size, electrophoretic mobility, or surface composition (phe-notype) (1). One of the earliest and still most commonly used methods is based on the use of centrifugal force to exploit density differences and deplete erythrocytes from whole blood. Further

From: Methods in Molecular Biology, vol. 295: Immunochemical Protocols, Third Edition. Edited by: R. Burns © Humana Press Inc., Totowa, NJ

refinements of this approach include the introduction of density gradients for increased resolution in cell density-based separations (2). This important advancement allowed routine isolation of blood mononuclear cell fraction for use in research (3). The use of modified silica coated with polyvinylpyrolidone (PVP) allows the use of continuous or discontinuous gradients, and these have been successfully applied in the density determination of several cell types, including leukocyte subsets (4). Cells isolated by these techniques can be authenticated by the use of antibodies that react specifically to surface markers, allowing the identification and quantification of cell types and subtypes.

It is necessary to take into account several factors when using density gradient centrifugation. Among these are the osmolality of the gradient, potential toxicity of the gradient, temperature, the effect of the gradient cushion on the cell population to be recovered, and cell concentration load (5). The cell density depends on the properties of the suspension medium, as well as the cell function. High medium osmolality (relative to the cell) can result in cell dehydration and subsequent changes in cell density. Additionally, when there is a mixture of cells in the suspension, osmolality effects may be different for each cell type and therefore bias the distribution of the cells separated by this process. Temperature affects the density as well; the lower the temperature, the higher the density of a liquid.

Cell suspension concentration effects have been studied empirically and theoretically optimized for several applications which, demonstrates it to be one of the most important parameters during density gradient centrifugation. Early studies on the hydrodynamics of particle sedimentation have shown the influence of particle size and density in centrifugation (6). For instance, high-density particles tend to produce a drag effect over low-density particles, pulling them to the high-density zone. This in turn decreases the purity of the high-density population and reduces the yield of the low-density population. Another important effect is the concentration of the cell suspension as the apparent viscosity of the solution increases with the increasing cell concentration (7).

Enrichment of rare cancer cells from peripheral blood samples is an application that typically requires density gradient centrifuga-tion as a first step. Application of this technique addresses two objectives: depletion of erythrocytes and depletion of polymorphonuclear cells. It is expected that cancer cells undergo sedimentation with the mononuclear cell fraction because of their similar density. However, some studies have found that cancer cells are also lost in the polymorphonuclear fraction or the erythrocyte fraction (8,9). Optimization of the density gradient sedimentation step is an important issue in such an application, because it will determine the recovery of rare cells from blood and affect the chances of their detection by immunochemical means.

The following section describes the steps necessary to isolate carcinoma cells (cell line, MCF-7) from a buffy coat. A filtration step is added at the end of the density gradient centrifugation procedure to concentrate cells onto a polycarbonate membrane, which is a convenient vehicle for analysis by immunocytochemistry.

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