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Different-Cell-Separation-Strategies

Different Cell Separation Strategies

richardjgray
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Different-Cell-Separation-Strategies

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  1. Different Cell Separation Strategies CD Why Do We Separate Cells? The separation of biological cells is definitely important to a variety of biomedical applications such as diagnostics, therapeutics and fundamental cell biology. Since samples of interest are usually heterogeneous populations of cells that are in culture or that comprise a tissue, mastering techniques to isolate specific cells are essential for understanding how cells function and respond to various stimuli. For example, blood is an extremely information-rich and easily accessible tissue which is a complex blend of cells. Thus, before accurate analysis of blood character and condition, it requires a separation of a few desired cells. White Blood Cells (7 - 9 × 106 cells/mL) Lymphocytes Monocytes Platelets (1.5 - 3 × 109 cells/mL) Hematopoietic Stem Cells (1000 - 10,000 cells/mL) Endothelial Progenitor Cells (100 - 1000 cells/mL) Circulating Endothelial Cells (10 - 100s cells/mL) Plasma Fibrocytes (10 - 100s cells/mL) Circulating Tumor Cells (0 - 1000s cells/mL) Buffy Coat Red Blood Cells ~ 5 × 109/mL Granulocytes (1.5 - 8 × 106/mL) Fetal Cells (0 - 300 cells/mL) Strategies for Cell Separation However, when cell size or density differences are not significant, effective cell separation is impeded by these techniques and other methods must be applied including fluorescence activated cell sorting (FACS) and magnetic activated cell sorting (MACS). In this context, magnetic particles (nanoparticles, 10 to 100 nm; sub-micron particles, 0.1 to 1 micron; microparticles, 1 to 50 microns) have been an important component of cell separation techniques in both biomedical research and in clinical medicine for the past decades. Tel: 1-631-624-4882 Email: info@cd-bioparticles.com

  2. There are numerous cell separation techniques that have been developed for benchtop and clinical setting. These techniques are based on either physical properties of the cell (such as density or size) or on cell affinity (depending on electric, magnetic or adhesive properties specific to each cell type). Standard techniques for the separation of cells include processing steps of filtration, centrifugation and sedimentation, which are carried out either in a batch or in a continuous manner and can be easily translated to a large-scale operation. Strategies Key parameters Advantages Disadvantages - Adhesion and growth property differences generally comparable across cell types - Very low throughput (> 3-5 days) Culture-based Separation Cellular adhesion profile Label-free - Physical and biological differences can be too subtle - Cell perturbations due to physical forces Label-free Standardized Equipment High throughput (>1011 cells/hr) Cell size Cell cytoplasm density Centrifugation Contract Factor (cell density and compressibility) Label-free Gentle on cells - Most cells have contrast factors of the same sign Acoustophoresis - Biological basis underexplored - Potential differences can be too subtle - Low to medium throughput (107-108 cells/hr) Cell dielectric properties Cell size Electric field parameters Label-free Easy incorporation into devices Dielectrophoresis - Problems with clogging - Physical and biological differences can be too subtle - Damage to cells Cell size Cell shape Cell deformability Mechanical/ hydrodynamic No exogenous labeling High throughput (>1010 cells/hr) - Requires cell-specific marker - Dependent on antibody-ligand specificity - Lack of a standard detachment method Cell surface marker expression Antibody/ligand binding kinetics Cell interaction with surface Cell-affinity Chromatography Highly specific Fluorescence activated Cell Sorting (FACS) Gives spatially specific information Identifies complex/subtle phenotypes - Often requires exogenous labeling - Trade-off between speed and resolution - Low throughput 107 cells/hr Optical signal intensity or morphological features Magnet activated Cell Sorting (MACS) Magnetic field strength Cell surface marker expression Magnetic label binding kinetics Can be highly specific Easy incorporation into devices - Often requires exogenous labeling - Medium throughput 109 cells/hr Tel: 1-631-624-4882 Email: info@cd-bioparticles.com

  3. Why Choose Magnetic Cell Sorting? Compared to other cell enrichment methodologies, combing magnetic forces with biochemically-labeled magnetic nanoparticles is an ideal approach to guide cell motion in a sample. This approach possesses the following advantages, making it especially suitable for targeted rare cell separation. A C Selectivity Throughput Similar to adhesion-based approaches, magnet-based separations have good sensitivity that arises from robust antibody–antigen binding between the cell and the magnetic particle label In comparison to cell-affinity chromatography, where direct contact between cells and surface molecules is essential for successful cell capture, magnetic assays can attract cells over a wider spatial domain E Integration A magnetic field acts at a distance and can be introduced without direct contact with cells. Further- more, the MACS separation platform can be integrated easily with other separation methods D B Specificity Tunability Using magnetic force as the retaining force, an immunomag- netic assay fosters good contrast between target and non-target cells in terms of the surface attachment. In the end, it is possible to remove the non-tar- get cells from the suspension, leaving behind enriched cells of interest Compared to techniques that feature a fixed filtration structure or surface mole- cule immobilization, the magnetic field component of MACS can be easily and accurately controlled, especially when an electro- magnet is used as the magnetic field source Tel: 1-631-624-4882 Email: info@cd-bioparticles.com

  4. Magnetic Cell Separation Strategies Single separations Magnetic labeling Magnetic separation Elution of the labeled cells S N Direct label √ Positive selection of a target cell type • Magnetically label and isolate target cell type from the cell suspension; • Require specific marker for the target cell type. N S S N Indirect label N S Magnetic labeling Magnetic separation √ Depletion of an unwanted cell type • Magnetically label and remove unwanted cell type from the cell suspension; • Require specific marker for the unwanted cell type. S N Magnetic labeling Magnetic separation √ Untouched isolation of a target cell type • Magnetically label and remove all unwanted cells from the cell suspension, leaving the target cells unlabeled (i.e., untouched); • No marker for target cells is required. S N Email: info@cd-bioparticles.com Tel: 1-631-624-4882

  5. Sequential separations A Magnetic labeling A Magnetic separation B Magnetic labeling B Magnetic separation Elution of the labeled cells S N S N N S √ Depletion followed by positive selection • When undesired cells that express the positive-selection marker can be depleted effectively, no specific marker for target cells is required. Magnetic Labeling Strategies Using Absolute Mag™ Magnetic Particles • Direct magnetic labeling Direct labeling with magnetic particles is the fastest way of magnetic labeling. Only one incubation step is required, possessing a minimal number of washing steps leading to minimizing cell loss. This approach is available for human, mouse, rat and non-human primate cells. • Indirect magnetic labeling There are two steps for indirect magnetic labeling using primary antibodies and Absolute Mag™ Magnetic Particles: 1) labeling the cells with a primary antibody directed against a cell surface marker; 2) magnetically labeling the cells with Absolute Mag™ Magnetic Particles which binds directly to the primary antibody or to a molecule that is conjugated to the primary antibody (such as immunoglobulin, biotin and fluorochromes). This approach is useful for isolating untouched target cells. Schematic Diagram Cell Labeled with Absolute Mag™ Magnetic Particles Cell Labeled with Anti-Immunoglobulin Absolute Mag™ Magnetic Particles Cell Labeled with Anti-Biotin Absolute Mag™ Magnetic Particles Cell Labeled with Anti-Fluorochrome Absolute Mag™ Magnetic Particles Tel: 1-631-624-4882 Email: info@cd-bioparticles.com

  6. Magnetic Cell Isolation Kits Creative Diagnostics provides fast and handy cell isolation kits that isolate various highly purified cell populations, including but not limited to B cells, T cells, stem cells and others form different biological suspensions. Key features of Magnetic Cell Isolation Kits include high binding capacity, high purities and recoveries, good compatibility and nontoxic. • High purity and recovery rates • Fully compatible with any downstream application • No non-specific labeling of target cells For more information, view our website: www.cd-bioparticles.com Tel: 1-631-624-4882 Email: info@cd-bioparticles.com Address: 45-1 Ramsey Road, Shirley, NY 11967, USA Fax: 1-631-938-8221 5

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