Procedures for Performing Magnetic Separation of Cells

Magnetic separation of cells is usually performed in one of the following formats:

1. Direct method. The affinity ligand is coupled to the magnetic particles, which are then added directly to the cell sample. During incubation the magnetic particles will bind the target cells which can be then recovered using a magnet.

2. Indirect method. Target cells are first sensitized with a suitable primary affinity ligand. After incubation, excess unbound affinity ligand is removed by washing the cells and then magnetic particles with an immobilized secondary affinity ligand with affinity for the first ligand are added. The magnetic particles will bind the target cells, which can then be recovered using a magnetic separator.

Another differentiation of magnetic separation techniques is based on the selection of the magnetically labelled cells.

1. Negative selection. Negative selection is a method by which a cellular subset is purified by removing all other cell types from the sample. Both the direct and indirect method are applied for negative selection. The advantage is that the purification process does not involve any direct contact of magnetic labels with the cells to be isolated.

2. Positive selection. The target cells are isolated from the cell suspension. Both the direct and indirect method can be used. The separated magnetically labelled cell complexes can be further characterized directly, but in many cases it is necessary to remove larger magnetic particles from the positively selected cells after their isolation.

3. Depletion of cells. Depletion is a method by which one or more unwanted cellular subsets is removed from a cell suspension. Both the direct and the indirect procedure can be applied for this purpose.

Immunomagnetic Separation

Immunomagnetic separation (IMS) is the most often used approach for the isolation of cells. Most often, a monoclonal antibody is used for IMS, but also polyclonal antibodies are used successfully. IMS can be performed in all the formats mentioned above.

In the direct method the appropriate antibody is coupled to the magnetic particles and colloids, which are then added directly to the sample. Ideally, the antibody should be oriented with its Fc part towards the magnetic particle so that the Fab region is pointing outwards from the particle. Several procedures are available for direct binding of antibodies (Table 2).

The indirect method is also used very often. The cell suspension is first incubated with primary antibodies which bind to the target cells. Not only purified primary antibodies have to be used, crude antibody preparations or serum can also be used. After incubation, the unbound antibodies are usually removed by washing. Then magnetic particles with immobilized secondary antibody are added to bind the labelled cells. Target cells - primary antibody complexes - can be also captured by protein A or protein G immobilized on magnetic carriers. Alternatively, biotinylated or fluorescein-labelled primary antibodies and magnetic particles with immobilized streptavidin or anti-fluorescein antibody are used to capture the target cells.

The indirect method is generally more efficient in removing target cells from a suspension because free antibodies will find their target antigen more easily

Table 2 Selected procedures for binding of antibodies on magnetic particles and colloids

Adsorption of antibodies on hydrophobic magnetic particles (especially those made of polystyrene)

Covalent binding of antibodies on activated magnetic particles (e.g. tosyl-activated), or on magnetic particles carrying appropriate functional groups (e.g. carboxy, amino, hydroxy, hydrazide) using standard immobilization procedures Immobilization of secondary antibodies (i.e. antibodies against primary antibodies) on magnetic particles followed by binding of primary antibodies

Immobilization of biotinylated antibodies on magnetic carriers with immobilized streptavidin Immobilization of antibodies on magnetic particles with immobilized protein A and protein G Immobilization of antibodies tagged with oligo dA on magnetic particles with immobilized oligo dT

Immobilization of antibodies on magnetic carriers with immobilized boronic acid derivative via their carbohydrate units on the Fc part than antibodies bound to magnetic particles. The indirect technique is recommended when the target cell has a low surface antigen density or a cocktail of monoclonal antibodies is used. The direct method is usually faster and requires less antibody than the indirect method. Also, the direct method is advantageous when one does not want to cover all antigen sites with antibody.

Typically, 95-99% viability and purity of the positively isolated cells can be achieved with a typical yield of 60-99%. Depletion efficiency often reaches 99.9% and leaves remaining cells untouched. Sequential depletions are markedly more efficient.

Magnetic particles usually do not have any negative effect on the viability of the attached cells. Many types of magnetic particles are usually compatible with subsequent analytical techniques such as flow cytometry, electron and fluorescence microscopy, polymerase chain reaction (PCR), fluorescence in situ hybridization (FISH) or cultivation in appropriate nutrient media. In some cases, however, it is necessary to remove larger immunomagnetic particles from the cells after their isolation. The detachment process can be performed in several ways (Table 3).

Incubation time for cell separation is usually 5-60 min while the binding of primary antibodies to secondary coated magnetic particles usually takes 30 min or less. In positive isolation, the purity of cells generally decreases with time, although the yield increases.

Nonspecific interactions of nontarget cells with hy-drophobic magnetic particles can be expected. These interactions can be partially eliminated using bovine or human serum albumin, casein and nonionic ten-sides such as Tween 20.

Magnetic Separations using other Labels

Antigens Antigens immobilized on magnetic particles can be used for the isolation of antibody expressing or antigen-specific cells. This approach has been successfully used for selection of antigen-specific hybridoma cells or human antibody-producing cell lines.

Lectins Lectins immobilized on magnetic carriers can interact with saccharide residues on the cell surfaces. A typical example of this approach is the application of immobilized Ulex europaeus I lectin which binds to terminal l-fucosyl residues present on the surface of human endothelial cells. Magnetic beads can be released from the isolated cells using a free competing sugar.

Oligosaccharides Oligosaccharides immobilized on magnetic particles can be used for the rapid isolation of specific lectin-expressing cells. Target cells bound to the magnetic particles can be released using a free competing saccharide structure.

Bacteriophage Salmonella-specific bacteriophage immobilized to a magnetic solid phase has been used for the separation and concentration of Salmonella from food materials.

Table 3 Selected typical procedures for detachment of cells after immunomagnetic separation

Incubation of rosetted cells overnight in cell culture medium with subsequent mechanical forces such as firm pipetting, flushing the suspension 5-10 times through a narrow-tipped pipette Trypsin, chymotrypsin and pronase have general applicability for proteolytic detachment of isolated cells

Detachment with a polyclonal antibody that reacts with the Fab fragments of primary monoclonal antibodies on magnetic beads. This principle is commercialized by Dynal, Norway (DETACHaBEAD) Using synthetic peptides which bind specifically to the antigen-binding site of primary antibodies (Baxter Healthcare, Deerfield, IL, USA) Antibodies immobilized via carbohydrate units on the Fc part to the magnetic particles with immobilized -B(OH)3 groups are dissociated with sorbitol

A complex primary antibody-DNA linker can be split enzymatically using DNase

Cryptosporidium oocysts were successfully released from the immunomagnetic particles by decreasing the pH of the suspension (adding HCl)

Erbium ions, ferritin and magnetoferritin have been used for magnetic labelling of both prokaryotic and eukaryotic cells. Magnetotactic bacteria can be introduced into granulocytes and monocytes by phagocytosis which enables their magnetic separation. Submicron magnetic particles of y-Fe2O3 adhere to the surface of Saccharomyces cerevisiae, making the cells magnetic and amenable to magnetic separation.

Magnetotactic bacteria, due to the presence of ferromagnetic material in their cells, can be magnetically separated without any labelling. Erythrocytes can be separated by the high gradient magnetic separation technique after conversion of diamagnetic eryth-rocytes containing oxyferrohaemoglobin into paramagnetic red blood cells by the oxidation of the iron atoms in the cell haemoglobin to the ferric state (methaemoglobin). Erythrocytes, infected by Plasmodium, contain paramagnetic hemozoin, that is a component of malarial pigment. The paramagnetic moment of hemozoin is of sufficient magnitude to enable the separation of malaria-infected (hemozoin-bearing) erythrocytes.

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