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