Going Their Separate Ways: A Profile of Products for Cell Separation

Date: September 13, 1999Cell Separation Products Magnetic Cell Separation Technologies that isolate rare cell types to high purity are essential to the cell biology researcher. Understanding cell developmental pathways becomes increasingly significant as diagnosis and treatment of disease turns more to the molecular level.1 This diagnosis of cell-related diseases requires methods for detection, isolation, and analysis of individual cells regardless of their frequency.2 The hematopoietic system has provided an easily accessible model for studying the processes that regulate cell growth. Accessibility isn't everything, however, as the most primitive cells and their progeny are present at relatively low levels. To further complicate matters, no unique, stable phenotype has been discovered to permit the activities of these cells to be assayed directly.3 Functional assays to study developmental pathways take weeks to complete, which could be the difference between a novel discovery and a "me too" paper. Schmatic of Cytovax Biotechnologies' glass beads interacting with unwanted cells In recent years several companies have been working on methods for increasing the purity and recovery of rare cell types. Preenrichment steps usually start with "debulking" of the sample (for example, whole blood) based on the differences in cell density or size followed by a more selective method that can yield higher numbers of purified cells (for example, stem cells, T-cell subsets, tumor cells). A balance must be maintained during cell separation because high purity can result in low yield. T-cell enrichment techniques are vital in determining the rate of HIV progression and transplant rejection.4 The success of gene therapy depends on the ability to understand what makes stem cells renewable.5 In this profile, LabConsumer examines several different cell separation techniques that lead to enrichment and purification of almost any cell. Separation of cells requires the removal of one cell type from another by physical means. Fortunately, cells differ in size and density, which has led to the use of density gradients for removal of mature red blood cells from hematopoietic cell suspensions. Cardinal Associates has developed one such medium: a modified ficoll-hypaque-based density gradient system, NIM2, which separates neutrophils and monocytes from animal blood after a 30-minute centrifugation. Neutrophils are purified to greater than 85 percent purity. Residual mononucleated cells can be lysed for removal. Additionally, Cardinal makes a continuous density gradient medium (NIM) that is used to separate human mononuclear cells and neutrophils from whole blood, again in 30 minutes. Cells recovered from this procedure are greater than 95 percent pure and viable. Glass beads with attached polyclonal antibodies Cell suspension with unwanted cells labelled with monoclonal antibodies Another type of density-based isolation medium is Lymphocyte Separation Medium by Mediatech. It is designed for the isolation of lymphocytes from diluted whole blood. Blood is layered on a low-viscosity solution that is either dextran or ficoll based and centrifuged to separate low-density from high-density cells. Several of the separation methods to be discussed rely on density gradient preenrichment techniques. Unlike the above media, which remove unwanted cells based on movement in a density gradient, BioErgonomics' PrepaCyte specifically agglutinates cells via surface antigen recognition. This one-step buffer rapidly removes erythrocytes, granulocytes, monocytes, and B lymphocytes from human peripheral blood, bone marrow, and umbilical cord blood through 1 x g sedimentation. T cells, natural killer (NK) cells, and rare populations such as CD34+ progenitor cells are isolated with high yields (>85 percent). Cells remaining in the supernatant retain viability and are ready for tissue culture or further purification procedures. Before use with PrepaCyte, samples should be heparinized (treated with an anticoagulant). An equal volume of PrepaCyte is added to the cells followed by gentle inversion for 30 minutes. Tubes are then incubated upright to facilitate agglutination and precipitation. The supernatant contains the desired cells. This buffer system is suitable only with human samples. Sigma-Aldrich, Life Technologies, and Amersham Pharmacia Biotech also offer a selection of density gradient media complementing their extensive catalogs. Centricoll (Sigma-Aldrich) consists of silica coated with polyvinylpyrrolidine. Centricoll forms a self-generated gradient after centrifugation for 15 minutes at 10,000 x g and is suited for the separation of living cells and viruses. Life Technologies makes gradient systems (Nycoprep and Lymphoprep) for the isolation of human blood monocytes and human or animal mononuclear cells. Lymphoprep is also available in prefilled tubes. In addition, Life Technologies also offers OptiPrep, a general-purpose density gradient medium for separation of a wide variety of cells, organelles, or viral particles. Amersham Pharmacia Biotech produces Percoll and Ficoll-Paque Plus for the separation of lymphocytes. Calibration of Percoll gradients can be simplified with Density Marker beads. The beads are of predetermined densities and are color-coded. BioDesign Inc. of New York has a different approach for cell separation as well as collection of cells from a bioreactor. CellMicroSieves permit the gentle and rapid isolation of cells, organelles, or cellular debris using a precision woven matrix of nylon filaments. CellMicroSieves have highly controlled pore sizes that can specifically trap cells and organelles. Pore sizes range from 5 to 200 µm, which facilitates separation of dissociated cells by size. Additionally, CellMicroSieves remove pieces of dissociated cells from tissues, permitting only single cells to pass. CellMicroSieves have rapid flow rates, which also permits their use as a prefilter and for the removal of cellular debris prior to cell preparation. Sieves are steam autoclavable with low protein binding and can be washed and reused. Lastly, CellMicroSieves are resistant to many caustic alkali and organic solvents. The great diversity of monoclonal antibodies has led to several antibody-based cell separation techniques. One such technique employs the use of immunocolumns such as the Cellect and Cellect Plus systems by Cytovax Biotechnologies. Cytovax makes several columns for the enrichment of human T cells and their subtypes. The process of negative selection (unwanted cells adhere to the column) removes human B lymphocytes. Serendipitously, a large portion of unwanted cells is removed due to nonspecific interactions. Enrichment of the cells of interest occurs in three steps. The first step involves the reconstitution of the column reagent (polyclonal antibodies and glass bead matrix) and addition to the column. Next, buffer is added to the column to set the flow rate. The cell suspension--which does not necessarily need to be preenriched through density gradient centrifugation--is added to the column at a predefined flow rate. (Too fast a flow rate could prevent adhesion of the unwanted cells and too slow could lead to nonspecific binding of wanted cells.) Buffer is added to the column for the elution of the desired cells. Using this method, greater than 95 percent of B cells are removed. Cytovax also has immunocolumns for the isolation of CD4+ and CD8+ T cell subsets for humans, mice, and rats, as well as for the isolation of mouse B cells and of human cytotropoblast cells from placentas. Pierce Chemical offers several kits for the isolation and enrichment of T cells and CD8+ or CD4+ cells from peripheral blood, spleen, thymus, lymph, or bone marrow. IsoCell kits use affinity columns to remove 95 percent of unwanted cells (B cells and monocytes). There are also columns for the purification of either human or mouse cells. R&D Systems crafts its own line of immunocolumns to isolate naïve and memory T-cell subsets from human, mouse, and rat cells through negative selection. Leukocyte suspensions are incubated with a monoclonal antibody cocktail. Once loaded onto the column, B cells and NK cells bind to anti-Ig coated glass beads through F(ab)-surface interactions. Monocytes adhere to Ig coated glass beads through Fc interactions. Enriched T cells pass through the column, are recovered in the sterile eluate, and are ready for tissue culture with less than 2 percent nonselected cells present. The immunocolumns come in a variety of sizes with a wide range of loading capacities for processing variable numbers of cells. Micra Scientific offers a new separation technology that takes advantage of both density gradient separation and antibody selection. Their Advanced Cell trap-Cell Enrichment System (ACT-CES) combines buoyant density suspension (BDS, proprietary to Dendreon Corp.) with Micra's Nonporous Microsphere Synthesis (NPS) technology to produce a separation technique that utilizes common centrifuges. NPS beads provide a uniform surface for the binding of antibodies. BDS has a well-defined isotonic osmolality such that cells less dense than BDS will collect at the interface after centrifugation. The cell mixture is loaded onto BDS and centrifuged for 30 minutes. The cells at the interface can be collected by decantation because an air lock is formed by constriction in the ACT tube. This unique design creates a barrier that prevents contamination of the desired cells at the interface from cells in the pellet. This technique is enhanced by the introduction of dense NPS carrier beads to the initial cell sample. The cells are premixed with an antibody cocktail of monoclonal antibodies that recognize the unwanted cells. After a wash step, the mix is incubated with dense GAM IgG NPS beads, which form a coating on the cells. The cells are layered onto the BDS and centrifuged in the ACT tubes. Again cells are collected from the interface. This procedure allows the negative selection of cells that need no additional processing (such as removing the antibody or beads). Positive selection techniques are also available. Micra ACT-CES kits are designed for the treatment of 100 ml cell suspensions including whole blood. NPS beads are additionally coated with streptavidin to take advantage of biotin-linked antibodies. This system has been used to enrich CD34+ cells, deplete T-cells, purge tumors, and enrich breast tumor cells. From One Cell Systems comes an assay system that isolates individual cells for analysis using a gel microdrop. One Cell Systems' Gel Microdrop (GMD) technology encapsulates cells in CelBioGel™, a biotinylated agarose matrix. Streptavidin and biotinylated capture antibodies against the protein of interest are added, and captured protein is detected by binding of a fluorescent reporter antibody. The fluorescence intensity of each GMD can be determined by fluorescence-activated cell sorting (FACS), and single cells can be recovered based on this intensity. The gel matrix surrounding each cell is comparable to an antibody-coated microtiter plate well; it keeps the protein of interest within the immediate cellular environment. GMDs are very small (20-50 µm in diameter) and allow diffusion of molecules as large as 900 kDa. The matrix acts as a selective net to keep target products.6 After GMD preparation using the CellSys 100™ Microdrop Maker (which can be leased from the company), 5-10 percent of the microdrops are occupied with a single cell. The agarose matrix is dissolved from the sorted cells and individual cells can be cloned for further use. The above assay has also been tailored for selection of cells that secrete a desired protein. Using this assay, screening for detectable levels of the secreted protein, and dilutional cloning to obtain the desired cells no longer take weeks but can be completed in a single day, according to One Cell Systems. In addition to selection based on secretion, One Cell also makes a system for cytotoxicity testing. This system can measure growth of a colony from a single cell. Again cells are encapsulated in agarose-like matrix, but they are exposed to antibiotics, cytotoxic reagents, or growth factors before being stained with a fluorescent marker. Cells are screened by FACS and those colonies resistant to applied agents are detected by their increased fluorescence intensity. With this method, 0.1-1 percent subpopulations of resistant cells can be detected. These colonies can be recovered for further analysis by removal of the agarose matrix. Bacterial, fungal, and mammalian cells can be assayed. Polyscience's magnetic cell sorting protocol Magnetic cell sorting takes immunoselection a step farther. Cells are incubated with an antibody specific for a particular cell that is covalently linked to a magnetic bead. Bound cells are placed in a magnetic field; unbound cells flow through the magnetic phase while the target cells are retained in the column. Once the column is removed, the target cells are eluted. There are two main strategies for isolating cells: positive selection or enrichment and negative selection or depletion. With positive selection, the target cells are labeled with the magnetic bead and are retained in the column. In negative selection, the target cells are the unwanted cells and the cells of interest elute with the flow-through. The method of choice will depend on the antibodies available and the subsequent use of the cells. Several companies have developed magnetic separation strategies based on the above premise. Miltenyi Biotech manufactures the MACS system. MACS superparamagnetic beads do not retain residual magnetism when removed from the magnetic field. MicroBeads are an average 50 nm in diameter and made of iron oxide and polysaccharide. The beads form a colloidal suspension--they remain dispersed due to the random molecular bombardment of Brownian motion7--and the MicroBeads can be used for positive selection of cells with frequencies as low as 10-8. MicroBeads are biodegradable, so labeled cells retain their function with positive selection. Removal of the bead is not necessary for further analysis. With MACS, cells are labeled using positive selection (unless an antibody is not available) for 15 minutes with the MicroBeads. The mixture is loaded on a column packed with a matrix of ferromagnetic spheres coated with a biopolymer for uniform flow-through.8 The column is placed in the magnetic field of a MACS separator and the flow-through contains the negative fraction. Removal of the magnet and flushing of the column elutes the positively selected cells. MACS technology can separate 105 or 1011 cells using the same technique with different-size columns with a typical purity of 95-99.9 percent and greater than 90 percent recovery. Cells eluted from the column are ready for cell culture, flow cytometry, PCR, fluorescent microscopy, or FISH. Miltenyi has optimized kits to isolate and separate cells using direct labeling (antibodies are coupled to the beads), and selection occurs in one step. Additionally, cells can be labeled indirectly. Cells are first incubated with a primary unconjugated, biotinylated, FITC, or phycoerythrin-conjugated (PE) antibody followed by magnetic labeling by using an antiimmunoglobin, streptavidin, anti-FITC, or anti-PE MicroBeads, respectively. Another company using magnetic separation is Polysciences. Magnetic cell separation is achieved using BioMag, a nonuniform solid support composed of 1 µm magnetic particles with a silane-coated iron oxide core. The nonuniform shape of BioMag particles provides an increased surface area that yields a higher binding capacity. BioMag particles are supplied covalently attached to several secondary antibodies or can be purchased with a particle surface that is coated with amine or carboxyl groups for the covalent attachment of proteins, glycoproteins, or other ligands. The particles can be used for direct or indirect labeling using positive or negative selection. When used for positive selection, magnetic particles can be removed by cell surface turnover during cell culture or by enzymatic digestion. Sorting is accomplished by adding 20-100 magnetic particles per cell based on the total cell population. Lymphocytes may be partially purified using density gradient separation. Beads are washed in sterile medium before being added to the cells for 20-30 minutes at 4°C. When the incubation tube is placed in a magnetic stand, the selected cells are attracted to the side of the tube. Polysciences' BioMag starter kit contains amine-terminated particles, a separator, and ultrapure glutaraldehyde for coupling of a protein to the beads. Unconjugated particles are sold separately, as are several secondary antibodies (for example, goat antihuman IgG, goat antirat IgG, goat antimouse IgG). Particles are also available conjugated to fluorescein, protein A, protein G, biotin, and streptavidin for indirect labeling. StemCell Technologies' immu-nomagnetic cell separation system, StemSep, uses negative selection to isolate cells. (Positive selection is an option--StemCell can be contacted for more information.) Using StemSep, unwanted cells are cross-linked to magnetic colloidal dextran iron particles (20 nm diameter) using tetrameric antibody complexes. The complex for labeling human cells consists of two murine IgG1 monoclonal antibodies held in tetrameric array by two rat antimouse IgG1 monoclonal antibodies. One murine antibody recognizes an antigen on the surface of the unwanted cell; the other recognizes dextran, thus cross-linking the magnetic dextran iron particle to the unwanted cell. Indirect labeling is used for the isolation of murine cells. Rosette of unwanted cell and RBCs formed by StemCell Technologies' RosetteSep™ Tetrametric Antibody Complexes Unwanted murine cells are first labeled with a cocktail of biotinylated antibodies directed against cell surface markers. The cells are then cross-linked to magnetic dextran iron particles using tetrameric antibody complexes that recognize biotin and dextran. To enrich human cells using StemSep, the sample is first incubated with a cocktail of tetrameric antibody complexes that label the unwanted cells, and then with the magnetic colloid. The sample is passed through a column of stainless steel mesh placed in a magnet, and the labeled cells are retained in the column. Murine cells are enriched in the same way, after an initial incubation with a cocktail of biotinylated antibodies. Greater than 99.9 percent of the labeled (unwanted) cells are retained in the column, enabling efficient enrichment of rare cells. The desired, unlabeled cells are collected in the flow-through and are ready for use in flow cytometry, cell culture, or functional assays. Numerous StemSep antibody cocktails are available to enrich hematopoietic progenitors, lymphocyte subsets, dendritic cells, baso-phils, monocytes, circulating epithelial tumor cells, and human cells from a murine/human chi-mera. Custom antibody cocktails are also an option. Five different columns, with capacities ranging from 1 x 105 to 1.5 x 1010 cells, can accommodate a wide range of sample sizes. Four magnets are available for use with the different columns, one of which can hold up to eight columns simultaneously. StemCell's newest product is the RosetteSep™ for obtaining highly purified cells from whole blood. Dynal has a vast array of magnetic particles for cell separation that can be used for either positive or negative selection. Dynabeads are superparamagnetic monodispersed polymer beads that have an even dispersion of magnetic material (gamma Fe2O3 and Fe3O4) throughout with a thin polymer shell. This provides bead uniformity and allows rapid interaction between the bead and the target. Beads come in three sizes (2.8, 4.5, and 5 µm) and are available coated or uncoated with antibodies. The desired application will determine the appropriate bead size. Dynabeads can be used to isolate cells from whole blood, bone marrow, buffy coat, or suspensions from prepared tissues. The products for separation fall into one of two categories: either ready to use (beads are targeted to common cell types) or for use with an antibody (beads are precoated with antibodies that recognize either mouse, rat, or rabbit primary antibodies). The only other equipment required is the Dynal MPC, which draws the beads to the side of the reaction tube. Positive selection of cells begins with incubating the starting sample for 15-30 minutes at 2-8°C. The suspension is then placed in the Dynal MPC and the cells are attracted to the side of the tube. The beads can be removed using DETACHaBEAD, a polyclonal anti-Fab antibody that is specific for the antibodies on the bead. DETACHaBEAD competes with the antigen/antibody complex at the cell surface, which leads to release of the bead and antibody from the cell. Additionally, with the CELLection positive isolation system a unique DNA linker between the antibody and the bead surface permits the cleavage of the DNA and detachment of the bead. Dynal's CELLection kits for positive selection are available for human T cells and for use with a mouse primary antibody or with biotinylated antibody. Its newest kits are for the negative isolation of human cells (monocytes, T cells, and B cells). Transiently transfected cells can also be identified using the Dynabead technology. The resulting cells from either positive or negative selection protocols are suitable for flow cytometry, cell culture, or assays to evaluate cell function and interactions. Magnetic particle separation products from SCIGEN are distributed through Vector Laboratories. The average particle size is 3.5 µm (sizes range from 1-10 µm), and the bead composition is magnetite and cellulose. The cellulose matrix provides a large surface area for efficient reactions. These beads can be coupled to any ligand for the isolation and separation of cells. Cortex Biochem offers a full range of magnetic particles that can be coupled to essentially any desired antibody, protein, enzyme, or substrate. The MagaPhase technology encapsulates ultrapure iron oxide in a variety of polymer supports such as polysaccharides, acrylic polymers, and various copolymers. MagaPhase has demonstrated applications in high-volume automated clinical analyzers. Contemporary applications of this product are under development for laboratory, industrial, and environmental scale biohazardous chemical, biological, and radioactive waste remediation. Extraction and stripping of hazardous materials from aqueous environments using MagaPhase and other proprietary resins have demonstrated their efficacy at U.S. Government Laboratory test sites and outside the United States. Several patents have been issued for Cortex Biochem's MagaBeads™, which are spherical magnetizable particles available amino- or carboxy-terminated for attachment of ligands, as well as streptavidin and A or G protein-coated. The resulting paramagnetic beads can be used in manual or automated assays for cell separation. CPG Inc.'s Magnetic Porous Glass (MPG) is a spheroidal porous glass embedded with superparamagnetic iron oxide. This magnetic particle offers the cell biologist and the molecular biologist the ability to have derivatized and underivatized magnetic particles with greater yield. MPG beads are 5 µm in diameter and may be coated with streptavidin. CPG also sells a three-in-one Magnetic Particle Separator that can hold single or multiple 1.5 ml microcentrifuge tubes or one 15 ml or 50 ml conical tubes. CPG's newest products for cell separation include its Protein A- and Protein G-coated porous glass particles. TECAN's Robotic Sample Processor customized for magnetic bead separation From Bangs Laboratories comes ProActive protein-coated microspheres for cell separation. ProActive beads can be streptavidin-coated and are superparamagnetic. Microspheres range from 20 nm to 1 mm in diameter and come in a variety of colors. Additionally, Bangs Labs manufactures classic magnetic microspheres (~1 µm diameter) derivatized with either -COOH or -NH2. These microspheres contain magnetite in a polymer matrix. Alexon Trend/Seradyn's entry is the Sera-Mag Magnetic Streptavidin. These particles are 1 µm beads that can be used to bind a variety of biotinylated ligands such as antibodies. Particles are available in a series of low to high biotin-binding capacities. Seradyn also makes Magnetic Carboxylate-Modified (MG-CM) in a 1µm diameter. The surface is modified with carboxylic acid groups for covalent coupling of proteins. These super-paramagnetic particles are uniform in size with quick reaction times and are stable in a pH range of 4 to 11. MG-CM can be used for all of the classic isolation needs such as cell separation. For those labs in need of an automated separation procedure, Tecan offers the GENESIS system. GENESIS is a robot sample processor that can be customized for the separation of magnetic beads. The system is capable of handling 24 test tubes, 48 microfuge tubes, or a 96-well PCR plate. The magnetic strips used for attracting the beads are mobile and pull the beads to the side of the tubes for easy aspiration of the supernatant and subsequent washing steps. Cell Separation Products Magnetic Cell Separation Michelle Vettese-Dadey (dadey@rcn.com) is a freelance science writer. M. Assenmacher et al., "Fluorescence-activated cytometry cell sorting based on immunological recognition," Clinical Biochemistry, 28:39-40, 1995. A. Radbruch et al., "Detection and isolation of rare cells," Current Opinion in Immunology, 7:270-3, 1995. T. Thomas et al., "Purification of hematopoietic stem cells for further biological study," A Companion to Methods in Immunology, 17:202-18, 1999. D. Collins et al., "T-lymphocyte functionality assessed by analysis of cytokine receptor expression, intracellular cytokine expression and femtomolar detection of cytokine secretion by quantitative flow cytometry," Cytometry, 33:249-55, 1998. P. Smaglik, "Problems and promise loom for stem cell gene therapy," The Scientist, 13[15]:14, July 19, 1999. J. Weaver et al., "Gel microdrop technology for rapid isolation of rare and high producer cells," Nature Medicine, 3:583-5, 1997. D. Recktenwald et al., "Magnetic cell sorting with colloidal superparamagnetic particles," In Cell Separation Methods and Applications, New York, Marcel Dekker Inc., 1996, pg. 153-73.

Going Their Separate Ways: A Profile of Products for Cell Separation

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