Download Separation

Separation of the immune
competent cells
8th seminar
Cell separation
Physical isolation of the cells of interest from a heterogeneous population
Differences in the physical , biological or immunological properties of the cells
are utilized to separate the cells
(differences in cell surface receptor expression is often available – there is a possibility to further
investigate the separated living cells )
physical – density, size
cell biological – adherence, phagocytosis, sensitivity to the
medium
immunological – antigen differences (surface!)
Characteristics of the separation:
• purity
• recovery, yield, loss
• viability of the cells
Separation
Base strategies:
positive separation – labeling and separation of the cells of interest
e.g. Labeling of a cell surface molecule (receptor!) by a fluorescent antibody.
The cells become affected both by the separation environment and the
antibodies bound to the receptors. The purity of the separation is generally
high.
negative separation – get rid of the labeled unwanted cells (depletion)
The cells become affected only by the separation environment This is the
preferred strategy in the functional examinations.
Peripheral blood is an easily accessible source for the separation of
human immune cells
Separation of the plasma from the cellular components:
Separation by filtration (simple membrane or holofiber „membrane”)
Pore diameter for plasma separation: 0.2 to 0.6μm
The different density parts of the anticoagulated blood is separated to three
parts in undisturbed tube:
bottom: sedimented red blood cells
top: cell free plasma
the intermediate layer is called „buffy coat” contains the leukocytes, platelets
The process can be accelerated by centrifugation
Apheresis (ancient greek ἀφαίρεσις) -“to take away”
Separating one particular component of the blood and
returning the remainder to the circulation
• Donor apheresis
• Therapeutic apheresis
Donor apheresis:
• Plasmapheresis – processed (e.g. IVIG) or fresh frozen plasma for immunodefficient
patients or in acute infections as passive immunization
• Plateletapheresis (trombocytapheresis) – concentrated platelets for inherited or
induced thrombocytopenia (infections, chemotherapy, irradiation) or in the case of
thrombocyte disfunction
• Red blood cells (erythrocytapheresis) - for patients with anemia (inherited or
internal/external blood loss by surgery or trauma)
• Leukapheresis – buffy coat, mainly for autotransplantation
• isolating and protecting the leukocytes before chemotherapy
• monocyte separation for dendritic cell therapy
• leukapheresis after the mobilization of bone marrow stem cells – for stem cell therapy
or bone marrow transplantation (autologous, allogeneic)
Therapeutic apheresis:
• Removing abnormal/disfunctional components of the blood (humoral or
cellular)
• Replacing a component with a healthy donor apheresis product
• Blood component alteration (ex vivo therapy)
• Leukapheresis – Extreme high leukocyte number can lead to hemostatic disorders in
leukemia (accompanying shortness of breath, vision changes). Inflammatory cell number can be
decreased in autoimmune chronic inflammatory diseases (ulcerative colitis, rheumatoid arthritis)
• LDL apheresis – Removal of low density lipoprotein from the plasma in patients with familial
hypercholesterolemia (adsorption with ApoB affinity column or precipitation with acetate)
• Thrombocytapheresis – In essential trombocythemia/e.trombocytosis (rare disease) the
disfunctional very high thrombocyte number (with thrombosis and bleedings) can be lowered
rapidly in the rare cases of life threatening emergency situations
• Erythrocytapheresis - Removing abnormal red blood cells in patients experiencing sickle
cell crisis (in sickle-cell anemia)
• Plasmaexchange – Removing/replacement the plasma with autoimmune antibodies in
various autoimmune diseases (combined with immunosuppression) (eg. Myasthenia gravis,
Guillain-Barré syndrome, lupus, Goodpasture syndrome, Antiphospholipid antibody syndrome,
Behcet syndrome, etc….)
• Immunoadsorption with protein A/G – removal of allo- and autoantibodies (in
autoimmune diseases, transplant rejection, hemophilia) by directing plasma through protein A or
G-agarose columns
Continuous Flow Apheresis Systems
Continuous Flow Centrifugation (CFC)
Cross section representation of an old
fashioned washable apheresis centrifuge bowl
Thrombocytes and thrombocyte free plasma can
be separated by appropriate CFC methods
plasma, red blood
cells, and buffy coat
production
by continuous flow
centrifugation
plasma
production by
filtration
& WBC
concentrated
platelet production
from pooled buffy
coats
Buffy coat contains too much
erythrocytes to investigate the
white blood cell part. Further
separation is needed.
Ficoll-Paque
(1.077g/ml)
(Nature Protocols http://www.nature.com/nprot/journal/v3/n6/images/nprot.2008.69-F1.jpg)
Ficoll-Paque: density based cell separation
peripheral blood
pipettig the „ring”
containing the
mononuclear cells to
a new tube
centrifugation
to get rid of ficoll
plasma
mononuclear
cells
(PBMC)
ficoll
pipetting cells on ficoll,
or pipetting ficoll under
the cells
neutrophyl
granulocytes
Red blood cells
separated cells
Ficoll separation combined with rosette formation can be
used for the depletion of unwanted cells
negative separation
Isolating or depleting adherent cells
(negative and positive separation)
Cheap, simple, but only for adharent cells.
Low purity and recovery.
Antibody ”panning”
(negative and positive separation)
coated antibodies
Complement mediated lysis
antibodies
complement
LYSIS
(negative separation)
(Red blood cells could be lysated in mild hypotonic
ammonium-chloride buffer without any pretreatment)
Simple magnetic cell separation
Phagocyte cells can uptake small iron particles.
These cells could be separated with a strong magnet.
Magnetic immunoseparation (MACS)
antigene specific antibody
paramagnetic
bead
MACS
Magnetic cell separation (MACS)
separation of labeled cells
(positive separation)
MAGNET
MAGNET
column
depleting or selecting
unlabeled cells
(negative separation)
Magnetic column
CliniMACS – closed system magnetic cell separator instruments
CliniMACS Plus
CliniMACS Prodigy
MicroBeads are very small, usually don’t interfere
with cellular functions.
CD8+ T cells
„DETACHaBEAD”
Policlonal antibodies against the Fab fragment of the antigen specific antibodies can
be produced (including anti-idiotype antibodies). These could compete with the cell
surface antigens for binding to the „magnetic” antibodies. So the magnetic
antibodies could detach from the cells.
CD antigen
cell type
function
ligand
CD3
T cells
TCR signalling
-
CD4
helper T sejtek, (monocytes,
T cell coreceptor, (HIV
receptor)
MHC- II, HIV
pDC)
CD5
T cells, (B cell subset: B1)
adhesion, activation signals
CD72
CD8
cytotoxic T cells, (NK,  T cells)
T cell coreceptor
MHC I
CD14
monocytes, macrophages,
some granulocytes
LPS binding
LPS, LBP
CD19
B cells
part of the B cell coreceptor
CD28
T cells
costimulatory signals to T
cells
(B7-1, B7-2)
CD80, CD86
CD34
hematopoietic progenitor cell
adhesion
CD62L
(L-selektin)
(endothelial cells)
CD56
CD80, CD86
(B7-1, -2)
NK cell, (T and B cell subset)
homoadhesion (N-CAM
isoform)
APC: DC, B, monocyte,
macrophage
costimulatory signals
CD28, CD152
FACS
Example:
NKT cell separation (CD3/CD56)
NKT cells
NK cells
lymphocytes
Try to figure out how to achieve this by magnetic separation
The fluid stream break up into dropplets by the
vibration of the flow cell.
breakoff point
vibration (nozzle orifice of the flow cell)
+
+
+
+
+
+
+
+
+
Laser
+
charged deflection +
plate
+
+
If the wanted cell reach the
breakoff point, the stream
become charged for the
short time of drop formation,
and the formed drop become
charged
+
+ +
+ +
- charged deflection
plate
-
--- collection tube
collection tube
waste