ABSTRACT
Selective cell separation requires a method to identify the target cell and a means to effect its collection. In immunomagnetic separations, identification is achieved by use of a mAb directed against a cell-surface component that is not expressed by other cells in the mixture. This component need not, however, be biologically exclusive to the target cell population; for example, an Ab used for removing tumor cells from bone marrow may cross-react with Ags expressed on liver cells and still be suitable for the intended use. Other agents that bind to cell surface components have also been used, e.g., lectins (32,33); however, their specificity towards a particular cell type requires extensive confirmation if they are to achieve the performance that is characteristic of mAb. Once the target cell has been identified, it must be linked to the solid phase that will be used to separate it physically from the mixture. This consists of a superparamagnetic microsphere or bead that has been coated with an Ab that reacts with the mAb used to identify the target cell, i.e., an antiimmunoglobulin Ab. Alternatively, if biotinylated mAbs are used, an avidin-or streptavidin-coated bead can be used for capture (34). The coated microspheres are added to the cell mixture and mixed gently for 30-90 minutes, during which time the beads attach specifically to the target cells to form a rosette (Fig. 3). These rosettes, together with any remaining nonrosetted
Figure 3 Lymphoma cell surrounded by immunomagnetic microspheres. (Scanning electron micrograph provided by Dr. Gunnar Kvalheim, Norwegian Radium Hospital, Oslo.)
beads, can then be separated by exposing the cell mixture to a magnetic field, such as that generated by an array of permanent magnets. The nontarget cells remain in suspension and can be collected by aspiration or decanting, whereas the target cells and free beads are attracted to the magnet and are retained against the side of the separation vessel. The positively selected target cells can be recovered by removal of the magnetic field and resuspension of the rosetted beads in a suitable cell culture medium. Removal of the beads from the cells can be achieved in a number of ways, as discussed later.
