Separation and Expansion of
Human T Cells
For the characterization of specific cell types and
the investigation of their functions, it is essential that
the cells can be purified. Cell separation techniques
based on the use of antibody-coated magnetic beads,
e.g., Dynabeads (Ugelstad et al.
, 1980, 1994), are now
widely used in research and clinical laboratories. Specific
cells can, after binding to the magnetic beads, be
selected by the use of a magnet and, following brief
washing, high cell purity can be achieved. This technique
continues to encompass new fields for the selective
isolation of eukaryotic cells (Funderud et al.
Luxembourg et al.
, 1998; Marquez et al.
, 1998; Soltys et al.
, 1999; Chang et al.
, 2002). The use of pure cell populations
has also reached the field of therapy. Ex vivo
expansion and manipulation of isolated cells have
given promising possibilities in therapy, especially in
immunotherapy. Dynabeads, having approximately
the same size as eukaryotic cells, have proven to be
very efficient in the ex vivo activation of T cells, the
prime effectors of the acquired immune system
(Garlie et al.
, 1999; Lure et al.
, 2001). An ex vivoexpanded
population of T cells may be administrated
to the patient, thereby helping to fight diseases such as
cancer, HIV, and autoimmune disorders (Liebowitz et al.
, 1998; Levine et al.
, 1998, 2002; Thomas and June,
There are two main strategies for isolating a
specific cell type: a "positive selection" of cells of interest
or a "negative selection" where unwanted cells are
depleted (Fig. 1). By positive selection, a specific cellular
subset is isolated directly from a complex mixture
of cells based on the expression of a distinct surface antigen. The resulting immune complexes of beads
and target cells are collected using a magnet. By
negative selection, all unwanted cell types are removed
from the sample by the magnetic beads. Cells isolated
by negative selection have not been bound to antibodies
at any time. Surface antigen-bound antibodies may
elicit the transmission of signals across the cell membrane.
However, in general the purity obtained by
negative selection is lower than for positive selection.
It is important to note that Dynabeads can be used
directly in complex samples such as whole blood,
which offers rapid and direct access to the maximum
number of target cells that have undergone the
minimum amount of interference. Usually, Dynabeads
are precoated with target-specific antibodies (direct
technique). Alternatively, antibodies are first added
to the cell suspension and thereafter the labeled
cells are immobilized to Dynabeads through secondary
antibodies, e.g., pan antimouse IgG (indirect
The particular properties of the magnetic beads
make it possible to use positively selected cells directly
for cell stimulation or molecular studies, such as PCR
or RT-PCR, without the necessity of removing the
beads. However, in functional studies the beads
should be removed. For several cell types, Dynabeads
can be detached from the cells after isolation using a
polyclonal antibody (DETACHaBEAD) that binds to
the Fab-region of the cell-specific monoclonal antibody
(Rasmussen et al.
, 1992). No antibodies remain on the
isolated cells after detachment when using DETACHaBEAD
(Fig. 2). CELLection is another system for the
removal of beads from isolated cells. DNase is used
to cut a DNA linker between the beads and the antibodies,
removing the beads and leaving only the antibodies on the cell surface (Soltys et al.
Werther et al.
Factors such as incubation time, temperature, and
concentration of reactants have a measurable effect on
the efficiency of cell isolation using magnetic beads.
Furthermore, the process is also affected by specific
key parameters, such as the nature and state of the
target cell, characteristics of the antigen/antibody
binding, sample type, concentration, and ratio of beads
and cells. Successful cell isolation with Dynabeads,
which implies high yield and purity, is dependent on
the concentration of the magnetic beads, the ratio of
beads to target cells, and the choice of antibody.
Monoclonal antibodies are generally recommended
due to their high specificity toward the target antigen.
|FIGURE 1 Positive and negative isolation of cells using antibody-coated Dynabeads. By positive selection,
a specific cellular subset is isolated directly from a complex mixture of cells based on the expression of
a distinct surface antigen. The resulting immune complexes of beads and target cells are collected using a
magnet. By negative selection, all unwanted cell types are removed from the sample by the magnetic beads.
II. MATERIALS AND
|FIGURE 2 Detachment of cells from Dynabeads using DETACHaBEAD. Cells are detached from the
beads by a polyclonal antibody (DETACHaBEAD) that binds the Fab-region of the cell-specific monoclonal
antibody, thereby altering its affinity for the antigen. The cells are left without antibody on their surface.
Lymphoprep 6 × 500 ml (Prod. No. 1114547), density
1.077 ± 0.001g/ml, osmolality 290 ± 15mOsm from Axis-Shield Poc is stable for 3 years if stored at room
temperature in the dark. Human serum, AB 100ml
(Part Code US14-490E) from BioWhittaker is stable for
3 years if stored at -20°C. Human serum, AB should
be heat inactivated (56°C, 30min) before use. All
human blood-based products should be handled in
accordance with currently acceptable biosafety practices
and guidelines for the prevention of blood-borne
viral infections. Fetal bovine serum, premium, US
origin 500ml (Part Code US14-501F) from BioWhittaker
is stable for 3 years if stored at -20°C. Fetal
bovine serum should be heat inactivated (56°C, 30 min)
before use. Bovine serum albumin (BSA) >96% by electrophoresis,
500g (Prod. No. A-4503) is from Sigma.
DPBS, without Ca2+
500ml (Part Code BE17-
512F) from BioWhittaker is stable for 2 years if stored
at 15-30°C. RPMI 1640, with L-glutamine 500ml (Part
Code BE12-702F) from BioWhittaker is stable for 2
years if stored at 15-30°C. Proleukin interleukin-2
(recombinant IL-2) 22 × 106
IU from Chiron B.V. was
reconstituted in sterile H2
O at 6 × 105
IU/ml and stored
at -20°C for up to 1 year. CD4-FITC 100 tests/2 ml (Prod. No. 3021) from Diatec is a mouse monoclonal
IgG2a/κ, clone EDU-2 recommended for flow cytometry.
CD4-FITC should be stored at 2-8°C (short term)
or -20°C (long term). CD25-PE 100 tests (Cat. No.
341010) from BD Biosciences Pharmingen is a mouse
monoclonal IgG1, clone 2A3 recommended for flow
cytometry. CD25-PE should be stored at 2-8°C. CD8-
PC5 100 tests/2ml (Part No. IM2638) from Beckman
Coulter is a mouse monoclonal IgG1, clone B9.11
recommended for flow cytometry. CD8-PC5 should be
stored at 2-8°C. Dynal CD4 positive isolation kit, 2 ml
(Prod. No. 113.03), Dynabeads CD25, 2ml (Prod. No.
111.33), DETACHaBEAD CD4/CD8, 5ml (Prod. No.
125.04), Dynal CD4 negative isolation kit, 5 ml (Prod.
No. 113.17), Dynal CD8 negative isolation kit, 5ml
(Prod. No. 113.19), Dynabeads TcapTM
(Prod. No. 103.01), and Dynabeads CD3/CD28 T-cell
expander, 2ml (Prod. No. 111.31) are from Dynal
Biotech. These products, which contain magnetic
beads (2.8-4.5µm) and/or antibody cocktails (mouse
monoclonal antibodies) and/or release agents (polyclonal
sheep antimouse antibodies), are stable for
12-36 months when stored at 2-8°C. Magnets: Dynal
MPC (Prod. No. 120.01, 120.20, and 120.21) and from
Dynal Biotech. Sodium citrate dihydrate pro analysis, >99% pure, l kg (Cat. No. 1.06448.1000) is produced
by Merck and sold by VWR International. rHLAA2/
GLC-PE MHC tetramers 50 tests (code T2A-G)
from ProImmune are stable for >6 months when stored
at 2-8°C. Centrifuge: Rotanta 460 R from Hettich was
delivered by Nerliens. Sample mixer: Dynal MX1
(Prod. No. 159.07) with 12-tube mixing wheel (Prod.
No. 159.03) is from Dynal Biotech ASA. Flow cytometer:
BD LSR II with 488- and 633-nm lasers from BD
Biosciences was delivered by Laborel. Laminar flow
bench: Biowizard Kojair KR 200 from Kojair Tech Oy
was delivered by Houm AS. 37°C CO2
Forma Scientific Model 3548 (water-jacked incubator)
was delivered by Houm AS. Pipettes: Finnpipette
0.5-10 µl, 5-50 µl, 20-200 µl, and 200-1000 µl from
Thermo Labsystem were delivered by VWR
This article covers procedures for some basic principles
of cell isolation and cell stimulation using
The isolation of CD4+
regulatory T cells
(Shevach, 2002) demonstrates the different isolation
techniques. The isolation is performed in two steps; the
first step involves isolation of CD4+
cells by positive selection (protocol A) or negative selection (protocol
B). The second step involves isolation of CD25+
from the CD4+
cell population (protocol C).
Isolation of antigen-specific CD8+
T cells using
recombinant HLA molecules on Dynabeads demonstrates
enrichment of rare cells from a complex cell
sample (protocol D). Expansion of these antigen-specific
T cells demonstrates ex vivo
cell stimulation and
manipulation (protocol E). For further questions,
contact the supplier at: email@example.com.
A. Positive Selection of CD4+ T Cells
T cells are isolated from buffy coat by positive
selection using the Dynal CD4 positive isolation kit.
The isolated CD4+
cells are then ready for further
studies, e.g., isolation of the CD4+
subpopulation (protocol C).
Steps for Cell Isolation
- Phosphate-buffered saline (PBS) pH 7.4: Dulbecco's
PBS without Ca2+ and Mg2+
- PBS/citrate: PBS with 0.6% (w/v) sodium citrate (to
- Heat-inactivated fetal calf serum (FCS)
- PBS/BSA or PBS/FCS: PBS with 0.1% (w/v) bovine
serum albumin or PBS with 2% (v/v) FCS.
- Culture medium: RPMI 1640 with 1% (v/v) FCS.
Steps for Removal of Dynabeads from Cells
(D E TA CHa B EAD)
- Dynabeads required: 2 × 107 beads/ml × 40ml = 8× 108 beads (2.0 ml).
- Wash Dynabeads; mix vial, transfer beads to a
50-ml tube, add 10ml PBS/BSA, collect beads on a
magnet for I min, remove supernatant, and replace
with 2ml PBS/BSA; cool to 2-8°C.
- Dilute 15ml buffy coat in 25ml PBS/citrate; cool to
- Add 40ml of cell suspension to the Dynabeads
(2ml) directly into the 50-ml tube. Mix gently by
tilting and rotation for 30min at 2-8°C.
- Isolate the cells that are attached to beads (rosetted
cells) by placing the tube in the magnet for 2min.
Discard the supernatant while the rosetted cells are
held at the tube wall by the magnet.
- Wash cells; remove the tube from the magnet and
resuspend the rosetted cells gently in 10ml
PBS/citrate. Repeat steps 5 and 6 four to five
- Resuspend the rosetted cells in 1 ml of culture
medium in a 5-ml tube. The cells are now ready for
the removal of beads.
B. Negative Selection of CD4+ T Cells
- Add 10 µl DETACHaBEAD per 107-108 beads used
for cell capture.
- Mix gently by tilting and rotation for 45-60min at
- Pipette gently five to six times to resuspend the
cell/beads suspension and place the tube in a
magnet for 2min.
- Collect the supernatant while the beads are held at
the tube wall by the magnet.
- To obtain residual cells, resuspend the beads in
culture medium and repeat steps 3 and 4 twice.
- Combine the supernatants, and wash the cells twice
with PBS/BSA to remove DETACHaBEAD.
- The cells are now ready for further analysis or isolation
of subpopulations (protocol C).
T cells are isolated from peripheral blood
mononuclear cell (PBMC) by negative selection using
the Dynal CD4 negative isolation kit. Unwanted cells
are removed, and the CD4+
T cells are then ready for
further studies, e.g., isolation of the CD4+
T-cell subpopulation (protocol C).
PBS/citrate, PBS/BSA, FCS, and culture medium
are prepared as in protocol A.
Steps for Cell Isolation
C. Positive Selection of CD4+CD25+ Regulatory T Cells (Fig. 3)
- Dynabeads required" 4 beads/PBMC × 108 PBMC =
4 × 108 beads (1.0ml).
- Wash Dynabeads as described in protocol A, resuspend
in 1 ml.
- Isolate PBMC with low platelet content; add 35 ml
solution (10ml buffy coat +25ml of PBS/citrate) on
top of 15 ml of Lymphoprep at room temperature.
Centrifuge for 20min at 160g at 20°C. Remove 20 ml
of supernatant by suction to eliminate platelets.
Centrifuge for 20min at 350g at 20°C. Recover
PBMC from the plasma/Lymphoprep interface.
Wash PBMC three times in PBS/BSA (centrifuge for
8min at 500g the first time and then at 225g) and
resuspend the PBMC at 107 cells per 100-200 µl in
PBS/BSA. Keep cells at 2-8°C.
- Incubate 108 PBMC with the 200-µl antibody mix
and 200µl FCS for 10min at 2-8°C.
- Wash cells; add 5-10ml PBS/BSA and centrifuge for
8min at 500g at 2-8°C. Resuspend cells in 9ml
PBS/BSA at room temperature.
- Add 9ml of PBMC to the depletion Dynabeads
(1 ml) directly into the 50-ml tube. Mix gently by
tilting and rotation for 15 min at room temperature.
- Pipette gently five to six times to resuspend the
rosetted cells and double the sample volume with
PBS/BSA. Place the tube in a magnet for 2min.
Collect the supernatant containing CD4+ T cells. The
cells are now ready for further analysis or isolation
of subpopulations (protocol C).
T cells have been isolated by either positive
(protocol A) or negative (protocol B) selection. From
these cells the CD4+
regulatory T-cell subpopulation
can be isolated using Dynabeads CD25, and the
beads are removed with DETACHaBEAD.
|FIGURE 3 Isolation of CD4+CD25+ regulatory T cells.
CD4+CD25+ regulatory T cells were isolated according to protocol C.
Diatec CD4-FITC and Pharmingen CD25-PE (clone 2A3) were used
for cell staining. (A) Staining of CD25+ T-cells in the CD4+ T-cell
population. (B) Staining of CD25+ T-cells after positive isolation from
the CD4+ T-cell population.
Steps for Cell Isolation
- PBS, PBS/BSA, FCS, and culture medium are
prepared as in protocol A
- PBS/10% FCS: PBS with 10% (v/v) FCS
Steps for Removal of Dynabeads from cells
- Dynabeads required: 4 beads/CD4+ cell × 5 × 107 CD4+ cells = 2 × 108 beads (500 µl).
- Wash Dynabeads as described in protocol A, but in
a 15-ml tube, resuspend in 500µl.
- Prepare cells by protocol A or B, resuspend at 107 cells/ml in PBS/BSA, and cool to 2-8°C.
- Add 5 ml of cells to the Dynabeads (500µl) directly
into the 15-ml tube. Mix gently by tilting and rotation
for 20rain at 2-8°C.
- Isolate and wash the rosetted cells as described in
protocol A (steps 5 and 6), but use 2 ml PBS/BSA for
- Use precoated tubes (10min at room temperature
using PBS/10% FCS) from this step to avoid cell
loss. Resuspend the rosetted cells in 200 µl of culture
medium in a 1.5-ml tube. The cells are now ready
for removal of beads.
D. Isolation of Antigen-Specific CD8+ T Cells
- Add 10 µl DETACHaBEAD per 107 beads used for
- Incubate, resuspend, collect, and wash cells as
described in protocol A; use precoated tubes. The
cells are now ready for further analysis and functional
Dynabeads coupled with recombinant HLA (rHLA)
class I molecules loaded with the relevant peptide
can be used to enrich antigen-specific CD8+
(Garboczi et al.
, 1992; Luxembourg et al.
, 1998; Ostergaard
Pedersen et al.
, 2001). The starting material is
PBMC or preferably negatively isolated CD8+
(protocol B, using Dynal CD8 negative isolation kit).
T cells are isolated using
|FIGURE 4 Isolation of rHLA-A2/GLC positive T cells from
PBMC. (A) PBMC from a HLA-A2/GLC positive donor stained
with rHLA-A2/GLC tetramers. (B) PBMC after removal of rHLAA2/
GLC tetramer positive T cells by rHLA-A2/GLC coated Dynabeads,
i.e., Dynabeads HLA-A2 EBV/BMLF-1 (see protocol D). The
rHLA-A2 was produced and loaded with the peptide GLCTLVAML
according to Ostergaard Pedersen et al. (2001).
Steps for Cell Isolation
- PBS/BSA and FCS are prepared as in protocol A.
- Culture medium: RPMI 1640 with 5% (v/v) FCS or
5% (v/v) AB serum.
E. Activation and Expansion of T Cells
- Dynabeads required: 107 beads/ml × 1 ml = 107 beads (25 µl).
- Wash Dynabeads; mix vial, transfer beads to a 1.5-
ml tube, add 0.5ml PBS/BSA, collect beads on a
magnet for 1 rain, remove supernatant, and replace
with 100µl PBS/BSA; cool to 2-8°C.
- Prepare cells by protocol B (preferably negatively
isolated CD8+ T cells), resuspend at 5 × 107 cells/ml
in PBS/BSA, and cool at 2-8°C.
- Add 1 ml of cells to the Dynabeads directly into the
1.5-ml tube. Mix gently by tilting and rotation for
30min at 2-8°C.
- Isolate and wash the rosetted cells at 2-8°C as
described in protocol A (steps 5 and 6), but use 1 ml
PBS/BSA for washing. Resuspend the rosetted cells
in 100µl of culture medium (2-8°C). The cells are
now ready for further analysis or expansion (protocol
T-cell activation in vivo
is initiated by the binding of
T-cell receptors on its surface to appropriate peptide-
HLA molecules on the surface of antigen-presenting
cells (APC). However, activation of highly pure T cells in vitro
is difficult to perform without the presence of
APC. The Dynabeads CD3/CD28 T-cell expander can
mimic the APC and therefore be used to activate pure
T cells. Some T cells are CD28 negative and will require
other costimulatory signals than through CD28, e.g.,
through CD137 (Maus et al.
Steps for in vitro Cell Expansion
- FCS is prepared as in protocol A.
- Culture medium: RPMI 1640 with 5% (v/v) FCS or
5% (v/v) AB serum
- Culture medium/rlL-2: culture medium with
20-100 IU/ml recombinant IL-2
- Dynabeads required: 4 beads/T cell × 107 cells =
4 × 107 beads (1.0ml).
- Starting material may be monocyte-depleted
PBMC, pure CD3+ CD4+ or CD8+ T cells (e.g., prepared
according to protocol A or B). Resuspend at 106 cells/ml in culture medium and add 1 ml of this cell suspension to each well in a 24-well culture plate. If
rare cells are isolated and beads are still attached to the
cells (e.g., protocol D), each well should contain a
maximum of 106 beads (used for isolation), regardless
of the number of cells isolated.
- Add the Dynabeads CD3/CD28 T-cell expander
directly to the cells at a ratio of four beads per cell. Mix gently by pipetting.
- Incubate cells for 3 days at 37°C, resuspend, and
count the cells. Dilute to 0.5 × 106 cells/ml with culture
medium/rIL-2. Split cells every second to third day
with culture medium/rIL-2 to keep the cell concentration
at 0.5-1 × 106 cells/ml.
- Cells can be expanded for 14 days without
adding extra beads, and cell expansion rates are
usually between 100- and 1000-fold, depending on
Immunomagnetic cell isolation offers rapid and
direct access to target cells from whole blood and bone
marrow without cell loss or damage. If necessary,
unwanted elements such as erythrocytes, free DNA,
fat, or serum proteins may be removed before cell isolation
to improve the performance of the beads. Buffy
coat, a concentrate of white blood cells, has the advantage
of high target cell concentration. However, the
quality of buffy coat preparations may vary considerably.
Density gradient isolation of cells provides
removal of possible interfering elements and the
ability to manipulate target cell concentration to
perform cost-efficient cell isolation. Drawbacks
include cell losses during centrifugations and negative
effects on the cells due to contact with the density gradient
medium. Generally the concentration of nucleated
cells should be 108 cells/ml when performing
immunomagnetic cell isolation.
By positive selection the cells of interest are isolated
for analysis. General isolation parameters are ≥1 × 107
beads/ml, bead:cell ratio 4:1-10:1, and 10-30min of
incubation. Typically, 95-100% purity and viability are
achieved with 60-95% yield. For some downstream
applications the beads can remain attached to the cells
(e.g., mRNA or DNA isolation). By negative selection
), unwanted cells are removed prior to
analysis of the remaining population. General isolation
parameters are ≥2 × 107
beads/ml, bead:cell ratio
≥4:1, and 20-60min of incubation. Typically, 95-99%
depletion of unwanted cells is achieved. Two successive
depletion cycles may result in higher purity for
small cell populations. The direct technique offers fast cell isolation with antibody-coated beads. Cell handling
is minimized, reducing the risk for cell damage
and loss. The indirect technique is especially useful
when the affinity/avidity of the primary antibody is
low or when the epitope density on the target cell
is limited. The disadvantage of the indirect technique
is cell handling (centrifugation).
Cross-reactivity and Fc binding can be blocked by
the addition of free proteins (e.g., Fc receptor blocking
with γ-globulin). Nonspecific binding of "sticky" cells
to beads can be avoided by predepletion of these
cells with protein-coated beads (e.g., secondary coated
or BSA-coated beads). Free DNA will contribute
to binding of unwanted cells to the beads.
Freezing/thawing may damage cells and release free
DNA. DNase treatment prior to cell selection will
abolish this problem. Platelets may also induce nonspecific
binding of cells to Dynabeads. Preparation of
PBMC with a low platelet content (see protocol B) will
reduce this problem.
Primary-coated Dynabeads are ready-to-use products
for a wide variety of cell surface markers. In addition,
secondary-coated Dynabeads offer an excellent
possibility to make beads with the reactivity of choice
using mouse, rat, or rabbit antibodies directly from
culture supernatant, ascites, or polyclonal sera
(without the need of purification). This is especially
useful when only small amounts of nonpurified antibody
are available. However, affinity-purified antibodies
- Prolonged incubation (>60min) and increased
bead concentration (>1 × 108 beads/ml)will rarely
improve the cell selection efficiency. However, nonspecific
binding may increase, damage of cells from
sheer forces of the beads may occur, and risk of cell
- A soluble form of cell surface antigens or other
serum components can reduce the efficiency of
immunomagnetic cell isolation. One or two washing
steps will overcome this problem.
- Nonspecific binding. Genomic DNA from lysed
cells (e.g., present in buffy coat, PBMC, or after freezing/
thawing of cells) will induce non-specific binding
of cells to beads. DNase treatment of the cell suspension
prior to cell selection will prevent this problem
without harming intact cells. Some sample tubes (e.g.,
glass or polystyrene) tend to bind cells nonspecifically,
which can be a major problem when working with
minor cell populations (e.g., rare, circulating tumour cells). Precoating of sample tubes with a protein solution
before use or the use of low-binding plastic tubes
- Phagocyte cells (e.g., monocytes) will bind and
engulf beads if incubation is performed at temperatures
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