Download Cell Separation Methods

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