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Cell Separation Methods Dr. Michael Rieger 2012 Why cell separation? • Heterogeneous mixture of specialised cell types in tissues • Studying a distict cell type Single cell analysis or purification of homogeneous cell type required Keep in mind: Population analyses represent only the average of all cells! Stem Cell Biology in Time-Lapse – Stem Cell Selfrenewal and Differentiation Group of Michael A. Rieger, PhD Low frequency of stem and progenitor cells in bone marrow ~1 in 100 Pro-NK NK cell Pro-T T cell Pro-B B cell CLP 1 in 105 LMPP SELF-RENEWAL LT-HSC CD127 + CD117 (lo) Sca-1 (lo) Dendritic cell Mast cell CD34 + CD135 (hi) ST-HSC BMCP MPP Gr (basophil) CD34 CD135 CD150 + CD48 - CD34 + CD135 (lo) CD150 CD48 - Gr (eosinophil) CD34 + CD135 + CD150 CD48 CD244 + GMP Gr (neutrophil) CD117 (c-Kit) +, Sca-1 + CMP CD127 CD34 + CD16/32 CD117 + Sca-1 - CD34 + CD16/32 + CD117 + Sca-1 - Monocyte Osteoclast Macrophage Erythrocytes MEP Megakaryocyte CD34 CD16/32 CD117 + Sca-1 - Platelets Lineage Marker negativ adapted from Rieger and Schroeder, 2007 Considering the appropriate technique Properties of cells: •Cell size •Density •Behaviour •Surface charge / Hydrophobic surface properties •Antigen status General considerations •Physiochemical or immunological characteristics of cells •Toxicity and stress of method for the cells •Potential contamination •Positive or negative selection •Density-based separation, MACS, FACS •Manual or automated systems Methods • Separation according to density (and size): – Gradient centrifugation • Separation according to adhesion - Adhesion to surfaces - Adhesion to sheep erythrocytes: E-rosette formation • Separation according to surface markers – Panning – Dynal beads / MACS – FACS Differential centrifugation separation • according to terminal velocity of particles • Stoke´s law • vt = 2R2(ps-p)a/(9µ) • vt is the terminal velocity of the particle, – R radius of the particle – a centrifugal acceleration of the centrifuge – µ the viscosity of the medium, – ps density of the particle – p density of the medium. Density gradient centrifugation separation according to density alone Example: • Separation of MNC via Ficoll-Paque: Density of MNC (lymphocytes and monocytes) is lower than FicollPaque and of erythrocytes and PMNL is higher. • Ficoll-Paque: density 1.077 g/ml – Ficoll 400: a neutral, highly branched, hydrophilic polymer of sucrose which dissolves readily in aqueous solution. • Recovery of MNC: 60 %, purity 95 %. • Different Ficoll-Paque for murine cells: Histopaque 1083 (density 1.083 g/ml) Isolation of human lymphocytes via Ficoll-Paque Plasma MNC: lymphocytes + monocytes Ficoll erythrocytes + PMNC MNC: mononuclear cells, PMNC: Polymorphnuclear cells Blood smear Separation of monocytes/macrophages from MNC via plastic adhesion Adherent macrophages in M-CSF culture Development of adherent macrophages in M-CSF culture from granulocyte-macrophage progenitors Rieger et al. SCIENCE 2009 Isolation of T cells by rosette formation with sheep erythrocytes Mediated by CD2 (T cell) and CD58 (erythrocyte) interaction Separation of aggregated cells from unbound lymphocytes by ficoll paque centrifugation rosettes are pelletted CD: Cluster of differentiation •Cell surface markers •According to an international convention (CD nomenclature committee) •Currently more than 300 CDs listed (in human) Examples for human antigens: CD45: pan hematopoietic cell marker CD3: T cell receptor CD4: T helper cell marker (binds MHCII) CD8: T killer cell marker (binds MHCI) CD19: B cell coreceptor CD34: Hematopoietic stem cell marker CD133: Prominin (new stem cell marker) Further improvement of purification by rosette formation -Advantage: fast, easy handling -Enrichment of population possible -Depletion of populations possible Dynal beads • Supra magnetic beads • Coated with antibodies or other relevant ligands for separation of cells or other biological materials or molecules • uncoated beads for self-coating Dynal beads 4.5 μm hydrophobic Dynabeads®: - primarily used for cell separation and cell stimulation. The size and magnetic susceptibility of Dynabeads® make them ideal for viscous samples such as whole blood, bone marrow and buffy coat. 4.5 μm slightly hydrophobic M-500 Dynabeads®: - ultra-smooth surface of these beads allows for gentle separation of organelles for electron microscopy. 2.8 μm Dynabeads® (hydrophobic M-280 and hydrophilic M-270): - are used for a wide variety of molecular manipulations, affinity isolations and bioassays, where the beads act as solid-phase during capture, handling and detection. 1 μm Dynabeads® (MyOne™): - increased surface area per unit weight compared to the larger beads. This high capacity, hydrophilic bead is designed for the in vitro diagnostics (IVD), high throughput, routine market. Additionally, these Dynabeads® can be used in a wide range of different molecular applications. MACS=magnetic-activated cell sorting - Magnetic labeling of cells by antibodies (50nm superparamagnetic particles) - Biodegradable microbeads -> no removal from cells required - Direct magnetic cell labeling or -Indirect labeling (anti-Ig, anti-biotin, Streptavidin, anti-fluorochrome - Positive selection: desired cell population is magnetically labeled and isolated as the retained cell fraction - Negative selection: Depletion of undesired cells. Non-target cells are magnetically labeled and depleted from the cell mixture. The flow through contains desired cell fraction MACS=magnetic-activated cell sorting Positive selection Cell depletion, negative selection MACS=magnetic-activated cell sorting Positive selection Pros Cell depletion, negative selection - Only one antibody is required (easy, cheap, fast) - No bound antibodies to the cells of interest - High purity of sorted cells - Purification of cell population without known antigens possible - Combination with subsequent positive selection possible Cons - Potential interference with biological function of antibody-bound antigen - Potential interference with biological function of antibody-bound antigen - Antigen expression must be unique to the cells of interest - Relatively inpure - Many antibodies necessary MACS separation unit Automated MACS separation MACS for clinical applications CliniMACS instrument – GMP conditions allow clinical application Dynal versus MACS beads Dynal beads MACS Size 1-5 µm 50 nm Handling Simple More complex protocols Price cheap More expensive Final fate of beads Must be removed by detachment step Can remain on the cell Positive selection High purity Only for specific cell types Negative selection high purity lower purity Cell loss higher low New Dynal innovation for debeading procedure Combination of different separation methods StemCell Technologies – Isolation of regulatory T cells FACS: Fluorescence-activated cell sorting • Analysis of surface marker expression • High throughput method with single cell resolution • relative and absolute quantification of signal strength • up to 15 different detection channels (colours) can be analysed simultaneously • Modern sorters: analysis of 100 000 cells per second, sorting speed 20 000 cells/sec • Technically demanding and expensive FACS machine diagram Example: Sorting of hematopoietic stem and progenitor cells LT-HSC ME progenitors ST-HSC GM progenitors Simultaneous sorting of four populations (LT-HSC, ST-HSC, MEP and GMP) Comparison FACS versus MACS FACS MACS Techn. Complexity High Low Purity High (>98%) Intermed. (90-98%) Specificity High High Negative selection Possible Possible (low purity) Positive selection Possible Possible Multi marker selection Possible Very limited Risk for bacterial contamination Intermediate Low Sorting for distinct expression levels possible Not possible Sortierung of cells with intracellular fluorescence (eg. eGFP) possible Not possible Simultaneous sorting of different populations Possible Very limited and not simultaneous Summary Choosing the appropriate cell separation method: • Subsequent application • Required purity • Required yield • Manipulation of sorted cell population (antigen stimulation?) • Knowledge/Experience • Costs