Introduction

Early experimentation on magnetic cell separation was conducted on phagocytic cells (1) and red blood cells (2). Currently, the literature devoted to magnetic cell separation has reached a few thousand publications and shows the diverse benefits that the technology has made to life sciences and clinical research during the past 15 yr

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

(3-5). Information on magnetic cell separation protocols is also maintained on commercial websites (Miltenyi Biotec, Nexell, Immunicon, Bangs Polysciences, Dynal, Cortex Biochem, Pharmingen, StemCell Technology, Molecular Probe, to name a few; see also Table 1). Some important sources of information on the development of clinical applications of magnetic separation are hematology meetings (such as those organized by American Society of Hematology and the International Society for Experimental Hematology) and cellular therapy meetings (International Society for Cellular Therapy).

Methodologies are typically based on high-gradient magnetic separators (6) or open gradient separators (7). The desired cell population is tagged with magnetic particles in the micrometer to submicrometer size range, such as Dynabeads (8) or MACS beads (9), respectively, with a considerable selection of other types of magnetic particles also available (10,11). The ever-expanding field of cell surface marker characterization by immunophenotyping, with the resulting discoveries of new clusters of differentiation (CD) molecules, provides a rich source of candidate cell markers for magnetic particle tagging (12) using monoclonal antibodies as linkers.

Negative cell separation, by depletion of the unwanted cell population tagged with magnetic beads, provides a relatively simple and effective method for purging bone marrow of residual cancer cells (both the tagged cells and the free beads in solution are removed by the application of the external magnetic field [13,14]). A drop in fractional cell concentration of unwanted cells by more than five orders of magnitude (more than "five-log" depletion) has been reported (4). Perhaps more demanding and technically difficult is positive cell separation, in which the desired cell population is tagged with the magnetic beads and separated. A complication of this method is that the beads remain with the enriched cell population and removal of them may be needed prior to the cells being used for further work. Additionally, the antibody binding to the cell surface receptor may lead to a cell signaling cascade and undesirable cell activation. The main advantage is the high selectivity of the separation defined by the specificity of the targeting antibody,

Table 1

Examples of Commercially Available Magnetic Particles for Cell Separationa

Table 1

Examples of Commercially Available Magnetic Particles for Cell Separationa

Particle

Diameter

Representative

trademark

Manufacturer

(Mm)

ligands

Website

Dynabead

Dynal Biotech, Oslo, Norway

4.5

Monoclonal antibodies

www.dynalbiotech.com

BioMag

Bangs Laboratories, IN (part of Polysciences Inc., Warrington, PA)

1

Monoclonal antibodies

www.bangslabs.com

MACS

Miltenyi GmbH, Bergisch Gladbach, Germany

0.05-0.1

Monoclonal antibodies

www.milenyibiotec.com

BD IMag

BD Biosciences, San Jose, CA

0.2

Monoclonal antibodies

www.bdbiosciences.com

Captivate

Molecular Probes, Eugene, OR (in association with Immunicon Corporation, Huntingdon Valley, PA)

0.2

Monoclonal antibodies

www.probes.com

EasySep

StemCell Technologies, Vancouver, BC, Canada

0.2

Tetrameric-antibody complex

www.stemcell.com

"This table is intended for illustration purposes. Additional information is available on the Internet, in particular, at the website, www.magneticmicrosphere.com.

"This table is intended for illustration purposes. Additional information is available on the Internet, in particular, at the website, www.magneticmicrosphere.com.

with the additional benefits of high depletion rates of unwanted cells. The enrichment rates (the ratio of the final to the initial fractional cell concentration) in excess of 100 (or "two-log"), with concomitant depletion rates of four order of magnitudes ("four-log") have been reported (9). Further increases in the desired cell fraction purity can be obtained by a combination of negative and positive separation steps.

An important part of magnetic cell separation is the determination of product purity and recovery (or yield) by cell cytometry, typically using the automated, differential cell counts by fluorescence-activated cell sorting (FACS; ref. 15). This requires additional cell labeling with fluorescent antibodies against cell surface marker epitopes not occupied by the targeting antibodies used for magnetic separation. Alternatively, a sandwich labeling method has been adapted in which the primary antibody used for cell targeting is conjugated to a fluorescent label, and the secondary antibody specific to epitopes on the primary antibody carries the magnetic particle (16). "Immuno-fluoro-magnetic" sandwich labeling is particularly well suited to the use of small, colloidal magnetic particles because they do not interfere with the optical properties of the labeled cells and therefore do not require removal prior to FACS analysis.

The advantages of using submicrometer magnetic particles for tagging include formation of stable suspensions and a relatively short reaction time of the antibody-particle complex with the target cell marker (9). They have been used for blood cell progenitor enrichment prior to transplantation in cancer patients, detection of rare cancer cells in circulating blood, detection of fetal cells in maternal blood, and for monitoring changes in cellular content of grafts used for cellular therapies ("graft engineering"; refs. 4,9). The advantages of the larger, micrometer-sized magnetic beads includes the use of small, inexpensive magnets that may be used in combination with standard laboratory tubes and simplicity of the separation process. Automated magnetic separators have made possible the expansion of cellular therapies to smaller clinical centers, and the exploration of new cellular therapy modalities.

A typical magnetic cell separation protocol includes a number of steps that can be grouped as follows: cell sample preparation, cell labeling and tagging, magnetic separation, cell product post-processing, and cell product characterization. The actual implementation depends on the type of cell sample and the type of magnetic tagging bead. It may require running a number of initial tests in order to achieve the optimal experimental conditions. Typical optimization parameters include target cell purity, recovery (or yield), percent viability and the speed of the overall process. As an example, a protocol for a positive selection of natural killer cells from human peripheral blood is described (17).

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