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Cell Separation Methods
Dr. Michael Rieger2012
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 ofMichael A. Rieger, PhD
Low frequency of stem and progenitor cells in bone marrow
CLP
CMP
LT-HSC ST-HSC MPP
Pro-T T cell
CD34 -CD135 -CD150 +CD48 -
CD34 +CD135 (lo)CD150 -CD48 -
CD127 -CD34 +CD16/32 -CD117 +Sca-1 -
CD34 +CD16/32 + CD117 +Sca-1 -
CD34 -CD16/32 -CD117 +Sca-1 -
GMP
MEP
Lineage Marker negativ
CD127 + CD117 (lo)Sca-1 (lo)
NK cell
Dendritic cell
Gr (neutrophil) Osteoclast
MacrophageMonocyte
Megakaryocyte
Erythrocytes
SELF-RENEWAL
Pro-B B cell
LMPP
CD34 +CD135 +CD150 -CD48 -CD244 +
CD117 (c-Kit) +, Sca-1 +
Platelets
CD34 +CD135 (hi)
Pro-NK
Gr (basophil)
BMCP
Mast cell
Gr (eosinophil)
adapted from Rieger and Schroeder, 2007
1 in 105
~1 in 100
Considering the appropriate technique
Properties of cells:
•Cell size
•Density
•Behaviour
•Surface charge / Hydrophobic surface properties
•Antigen status
•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
General considerations
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
Example:
• Separation of MNC via Ficoll-Paque: Density of MNC (lymphocytes and monocytes) is lower than Ficoll-Paque 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)
separation according to density alone
MNC: lymphocytes + monocytes
Isolation of human lymphocytes via Ficoll-Paque
Plasma
erythrocytes + PMNC
Ficoll
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 beads4.5 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 Cell depletion, negative selection
Pros
Cons
- Only one antibody is required (easy, cheap, fast)
- High purity of sorted cells
- No bound antibodies to the cells of interest
- Purification of cell population without known antigens possible
- Combination with subsequent positive selection possible
- Potential interference with biological function of antibody-bound antigen
- Antigen expression must be unique to the cells of interest
- Potential interference with biological function of antibody-bound antigen
- 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
High purityOnly for specific cell typesPositive selection
lower purityhigh purity Negative selection
50 nm1-5 µmSize
higherlowCell loss
Can remain on the cellMust be removed by detachment step
Final fate of beads
More expensivecheapPrice
More complex protocolsSimpleHandling
MACSDynal beads
New Dynal innovation for debeading procedure
StemCell Technologies – Isolation of regulatory T cells
Combination of different separation methods
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
ST-HSC
GM progenitors
ME progenitorsSimultaneous sorting of four populations (LT-HSC, ST-HSC, MEP and GMP)
Comparison FACS versus MACS
Very limited and not simultaneous
PossibleSimultaneous sorting of different populations
Not possiblepossibleSortierung of cells with intracellular fluorescence (eg. eGFP)
Not possiblepossibleSorting for distinct expression levels
LowIntermediateRisk for bacterial contamination
Very limitedPossibleMulti marker selection
PossiblePossiblePositive selection
Possible (low purity)PossibleNegative selection
HighHighSpecificity
Intermed. (90-98%)High (>98%)Purity
LowHighTechn. Complexity
MACSFACS
Summary
Choosing the appropriate cell separation method:
• Subsequent application
• Required purity
• Required yield
• Manipulation of sorted cell population (antigen stimulation?)
• Knowledge/Experience
• Costs
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