Cat. no. 402.03D
Rev. no. 004
002
Dynabeads® ClinExVivo™ CD3/CD28
Sterile Product
For Research Use Only
INDEX
1. INTENDED USE
2. PRODUCT DESCRIPTION
2.1
2.2
2.3
2.4
2.5
2.6
Principle of Isolation and Expansion
Materials Supplied
Storage and Stability
Additional Materials Required
Media Preparation
Important Information
3. PROTOCOLS
3.1 Dynabeads Washing Protocol
3.2 Starting Material
3.3 Separation and Expansion of Cryopreserved
Apheresis Cells
3.4 Separation and Expansion of Fresh
(non-cryopreserved) Apheresis Cells
3.5 Procedures Incorporating Gene Transduction
4. GENERAL INFORMATION
4.1
4.2
4.3
4.4
Certification
USA (Device Master File)
Technical Service
Precautions
5.1
5.2
5.3
5.4
Patents and Trademarks
Intellectual Property Disclaimer
Warranty
Limited Use Label License
5. WARNINGS AND LIMITATIONS
6. REFERENCES
1. INTENDED USE
Dynabeads ClinExVivo CD3/CD28 are intended for
separation (1, 2) and ex vivo expansion (1, 5) of
human T cells for cell-based clinical research.
2. PRODUCT DESCRIPTION
Dynabeads ClinExVivo CD3/CD28 are uniform,
superparamagnetic, sterile, non-pyrogenic polystyrene beads with affinity purified mouse anti-human
CD3 and CD28 monoclonal antibodies covalently
bound to the surface.
Dynabeads ClinExVivo CD3/CD28 are produced
according to cGMP under aseptic conditions in a fully
validated Class 100 clean room using gamma irradiated Dynabeads. All processes are validated.
Sterility and endotoxin tests are performed according to United States Pharmacopeia.
2.1 Principle of Isolation and Expansion
Dynabeads ClinExVivo CD3/CD28 offer a simple
method for isolation (1, 2), activation (3) and expansion (1, 5) of human T cells. Firstly, CD3+ T cells are
separated and concentrated from the apheresis product by magnetic cell separation using Dynabeads
ClinExVivo CD3/CD28 (1, 2). Following separation,
the CD3+ T cells are cultured in the presence of the
beads. By combining anti-CD3 and anti-CD28 antibodies on Dynabeads, the beads will provide both
the primary and co-stimulatory signals that are
required for activation and expansion of T cells (4).
The activated T cells have been shown to produce
IL2, GM-CSF, IFN-γ and TNF-� (1, 4, 5). T cells activated with these Dynabeads can be expanded 1001000 fold over a 9-14 day culture period (1).
The T cell expansion process can be scaled up using
a bioreactor-based process (5). It has been shown
that the T cell expansion protocol can be optimized
to include expansion of antigen-specific T cells (6).
2.2 Materials Supplied
• 10 ml of Dynabeads ClinExVivo CD3/CD28.
Supplied as a sterile suspension containing 4 x 108
Dynabeads/ml in phosphate buffered saline (PBS),
pH 7.4, w/0.1% human serum albumin (HSA).
2.3 Storage and Stability
When stored in unopened vials at 2-8°C, Dynabeads
ClinExVivo CD3/CD28 are stable until the expiry date
stated on the label.
Keep Dynabeads in liquid suspension during storage
and all handling steps, as drying will result in reduced performance.
Resuspend well before use.
2.4 Additional Materials Required
For clinical research procedures, the principal investigator is responsible for ensuring that use of all
procedures, reagents, and equipment follow applicable guidelines, standards and regulations. The
materials and equipment listed below are recom1
• L-glutamine, (Gibco)
••
•
••
HEPES
buffer,
(Gibco)
TM (Gibco)
L-glutamine,
O
pTmizer
T-Cell
Expansion SFM (Gibco) serum
® (Chiron)
free1xformulationdesignedtosupporttheculIL-2,
Proleukin
HEPES buffer, (Gibco)
tureandexpansionofhumanTcells(orequiva® (Chiron)
1-L Bags
(Baxter
Lifecell® or CellGenix Vuelife™)
•• lent).
IL-2,
Proleukin
® or CellGenix
® or
•
3-L
Culture
Bags
(Baxter
Lifecell
CellGenix
Vuelife™)
1-L Bags (Baxter
Lifecell
• L-glutamine,
(Gibco)
Vuelife™)
®
3-L Culture
(Baxter Lifecell or CellGenix
• HEPES
buffer,Bags
(Gibco)
• Vuelife™)
Sampling site ®coupler with female luer (Charter
• IL-2,
Proleukin (Chiron)
Medical)
Sampling
site coupler
with
luerVuelife™)
(Charter
® orfemale
CellGenix
•• 1-L
Bags (Baxter
Lifecell
• Medical)
Terumo TSCD® Sterile Tubing Welder
• 3-L Culture Bags (Baxter Lifecell® or CellGenix
40µm - 80µm
Filter (Pall)
SterileTransfusion
Tubing Welder
•• Vuelife™)
Terumo
TSCD®In-line
• 40µm
10-lead- 80µm
harness
sets (compatible
with Terumo
Filter
•• Sampling
site In-line
couplerTransfusion
with female
luer(Pall)
(Charter
SCD 312 welder), (Charter Medical)
• Medical)
10-lead harness sets (compatible with Terumo
• SCD
Hemostats
tube
clamp
312TSCD
welder),
(Charter
Medical)
® Sterile
Tubing
Welder
• Terumo
•• Hemostats
COBE 2991
Cell
Washer Disposable Set
tube
clampTransfusion
• 40µm - 80µm
In-line
Filter (Pall)
(COBE/Gambro) or Cytomate Disposable Set
•• 10-lead
COBE 2991
Cell
Washer
Disposable
Set
(Baxter) harness sets (compatible with Terumo
(COBE/Gambro)
Cytomate
Disposable Set
SCD
312 welder), or
(Charter
Medical)
• (Baxter)
Cell mixer (e.g. Heidolph Polymax 2040)
• Hemostats tube clamp
•• Cell
Plasma
device Polymax
(e.g. Thermogenesis
mixerthawing
(e.g. Heidolph
2040)
• COBE
MT202) 2991 Cell Washer Disposable Set
• (COBE/Gambro)
Plasma thawing or
device
(e.g. Disposable
Thermogenesis
Cytomate
Set
• MT202)
Bioreactor (e.g. Wave Bioreactor)
(Baxter)
Bioreactor
(e.g. Heidolph
Wave Bioreactor)
••2.5Cell
mixerPreparation
(e.g.
Polymax 2040)
Media
•2.5
Plasma
device (e.g. Thermogenesis
Buffer
1: thawing
Media
Preparation
MT202)
2+ and Mg2+, with 5% heat inactiPBS
without
Ca
Buffer 1:
•vated
Bioreactor
Wave
Bioreactor)
Pooled (e.g.
Human
Serum
(HS).
PBS without Ca2+ and Mg2+, with 5% heat inactivated
Pooled
Human
Serum
(HS).
2.5
Media Preparation
Incomplete
Medium:
Fig. 1: Schematic flow diagram for magnetic separation of CD3+ T cells with Dynabeads ClinExVivo
Fig. 1: Schematic flow diagram for magnetic
sepaCD3/CD28 and Dynal ClinExVivo™ MPC®
ration of CD3+ T cells with Dynabeads ClinExVivo
CD3/CD28 and Dynal ClinExVivo™ MPC®
mended for use with the Dynabeads ClinExVivo
CD3/CD28 procedures. Alternative materials and
Fig. 1: Schematic
for magnetic
sepamended
for use flow
with diagram
the Dynabeads
ClinExVivo
equipment may
be used.
+ T
cells with
Dynabeads
ClinExVivo
ration of CD3
CD3/CD28
procedures.
Alternative
materials
and
Materials
included, ™
butMPC
are®required to
CD3/CD28that
andare
Dynal
ClinExVivo
equipment
may
benot
used.
perform the procedures:
Materials that are not included, but are required to
mended
for procedures:
use
with the
Dynabeads
ClinExVivo
Epoxy
– Cat. no.ClinExVivo
402.01D
• Dynabeads
perform
the
CD3/CD28 procedures. Alternative materials and
MPC (Magnetic
Particle
concen• Dynabeads
Dynal ClinExVivo
ClinExVivo
Epoxy
–
Cat.
no.
402.01D
•equipment
may be used.
trator) Cat. no. 121.02
MPC
(Magnetic
Particle
concen•Materials
Dynal ClinExVivo
that are not included, but are required to
• trator)
Dynal MPC™-1
(Magnetic Particle concentrator)
Cat.
no. 121.02
perform
the
procedures:
Cat. no. 120-01D
Dynal MPC™-1
(Magnetic
Particle
ClinExVivo
Epoxy
– Cat. concentrator)
no. 402.01D
•• Dynabeads
• Cat.
PBS no.
(Gibco
) [Note: PBS must be calcium and
120-01D
ClinExVivo
• Dynal
magnesium
free.]MPC (Magnetic Particle concen• trator)
PBS (Gibco
) [Note:
Cat. no.
121.02PBS must be calcium and
• magnesium
Anti-coagulant
solution (e.g. ACD-A, Baxter)
free.]
• Dynal MPC™-1 (Magnetic Particle concentrator)
Pooled Human solution
Serum (e.g.
(HS) ACD-A,
(Cambrex
or Valley
•• Cat.
Anti-coagulant
Baxter)
no. 120-01D
Biomedical) heat inactivated at 56°C for 1 hour or
Pooled
Human
SerumPBS
(HS)must
(Cambrex
or Valley
be calcium
and
•• PBS
(Gibco
) [Note:
[Note: autologous
serum may
autologous
serum
Biomedical) free.]
heat inactivated at 56°C for 1 hour or
magnesium
be used, although significant donor to donor variautologous serum [Note: autologous serum may
ation should besolution
expected]
• Anti-coagulant
(e.g. ACD-A,
be used, although
significant
donor toBaxter)
donor variX-Vivoshould
15™
Medium,
(Cambrex)
•• Pooled
Human
(HS) (Cambrex or Valley
ation
be Serum
expected]
inactivated
at 56°C for 1 hour or
• Biomedical)
X-Vivo 15™ heat
Medium,
(Cambrex)
2 autologous serum [Note: autologous serum may
be used, although significant donor to donor vari2
ation should be expected]
Buffer
1: theMedium:
Combine
following components and filter
Incomplete
2+, protein
through
a sterile2+0.2
µmMg
low
binding
filter.
and
with 5%
heat
PBS
without
Combine theCa following
components
and inactifilter
vated
Pooled
Human
Serum
(HS).
•through
X-Vivo
15
or
equivalent
base
medium
with
a final
a sterile 0.2 µm low protein binding filter.
concentration of the following components:
• X-Vivo 15 or
equivalent base medium with a final
Incomplete
Medium:
• concentration
2 mM L-Glutamine.
of the following components:
TM
Combine
following
components
and serum
filter
OpTmizer the
T-Cell
Expansion
SFM (Gibco)
• 2
20mM
mML-Glutamine.
Hepes buffer.
•through
free1xformulationdesignedtosupporttheculture
a sterile 0.2 µm low protein binding filter.
• 20
5%mM
heatHepes
inactivated
Human Serum (HS).
andexpansionofhumanTcells(orequivalent).
buffer. Pooled
•• X-Vivo
15 or equivalent
base medium with a final
• concentration
5% heat inactivated
Pooled
Human
Serum (HS).
of the following components:
Complete Medium:
• 2 mM L-Glutamine.
Prepare fresh
Complete Medium every week by
Complete
Medium:
•adding
20 mM
200Hepes
IU/mLbuffer.
IL-2 to the Incomplete Medium.
Prepare fresh Complete Medium every week by
•adding
5% heat
inactivated
Pooled
Human
(HS).
Equilibrate
Complete
to
roomSerum
temperature
200 IU/mL IL-2Medium
to the Incomplete
Medium.
prior to use.
Equilibrate Medium:
Complete Medium to room temperature
Complete
prior to use.
2.6
Important
Information
Prepare fresh Complete
Medium every week by
adding
200
IU/mL
IL-2 to the Incomplete
Medium.
Follow
universal
precautions
when working
with
2.6
Important
Information
human serum,
plasma,Medium
or bloodtoproducts.
All human
Equilibrate
Complete
room
temperature
Follow universal precautions when working with
samples
must be treated as a potential source of
prior
to serum,
use.
human
plasma, or blood products. All human
HIV, HBV, and other blood borne pathogens. Gloves
samples must be treated as a potential source of
and a lab coat must
be worn when working with
2.6
Information
HIV, Important
HBV, and other
blood borne pathogens. Gloves
human samples.
Follow
universal
precautions
and a lab
coat must
be worn when
when working
working with
Materials
contaminated
with
blood
products
be
human
serum,
plasma, or
blood
products.
Allmust
human
samples.
decontaminated
bytreated
an approved
chemicalsource
method
samples
must
be
as
a
potential
of
Materials contaminated with blood products must be
and disposed
in labeled
biohazard
containers.
HIV,
HBV, and of
other
blood
borne
pathogens.
Gloves
decontaminated
by an
approved
chemical method
and
a lab coat
be worn
whencontainers.
working with
and disposed
of must
in labeled
biohazard
human samples.
Materials contaminated with blood products must be
decontaminated by an approved chemical method
Solution transfers that are not performed in a closed
system, such as spike connections and open containers, must be performed under a class 100 biological safety cabinet (BSC) using aseptic techniques.
The protocol below is specifically written to provide
guidance for activation (3) and expansion (1, 5) of
human T cells from cryopreserved apheresed products. Cultures may also be initiated from non-cryopreserved fresh samples, or samples derived from
sources other than apheresis, such as Ficoll separated whole blood (1, 3), cord blood (2) or bone marrow (7). As each sample source and method of T cell
or blood collection may vary, procedures may require specific modifications to maximize cell recovery, viability, activation and expansion. Such modifications must be determined empirically. Throughout
the protocol below, points to consider will be provided for possible protocol variations that may be
appropriate under certain experimental conditions,
such as gene modification or working with particular
disease tissues.
recommended that freshly collected samples are
cryopreserved and thawed prior to use.
[Cryopreservation and subsequent thawing facilitates lysis of granulocytes and other cell types that
can suppress T cell activation and expansion. This
may be particularly important when working with
samples from patients in certain disease states
where granulocyte counts are elevated.]
Alternatively, PBMC from freshly obtained leukapheresis product may be activated and expanded without cryopreservation. If fresh samples are used it is
recommended to deplete monocytes with Dynabeads (12, 13). We recommend to use Dynabeads
ClinExVivo Epoxy for monocyte depletion (see
Section 3.4)
For maximum activation and expansion of T cells in
PBMC, magnetically capture CD3+ T cells prior to
culture initiation (1, 2) (see Section 3.4.2). Magnetic
concentration is not required if T cells or T cell subsets have been enriched prior to activation and
expansion (e.g. purified CD4+ T cells) (8).
3. PROTOCOLS
3.3 Separation and Expansion of
Cryopreserved Apheresis Cells
3.1 Dynabeads Washing Protocol
3.3.1 Thawing and Washing of Cryopreserved
Apheresis Cells
Vials of Dynabeads ClinExVivo CD3/CD28 are at
negative pressure – liquid must be displaced with air
and vice versa.
2. Thaw the bag(s) in a plasma thawing device or by
equivalent methods.
Points to note are listed in brackets ([…]) throughout the protocol below. These identify areas where
modifications should be considered for specific circumstances.
Wash Dynabeads ClinExVivo CD3/CD28 twice before
use to avoid potential transfer of residual antibody.
1. Place one 10 ml vial of Dynabeads ClinExVivo
CD3/CD28 on the MPC-1 for 1 min.
2. Aseptically remove the supernatant.
3. Remove the vial from the MPC-1.
4. Aseptically add 10 ml Buffer 1 to the vial and mix.
5. Place the vial on the MPC-1 for 1 min.
6. Aseptically remove the supernatant.
7. Repeat steps 4 to 6.
8. Resuspend the beads in 10 ml of Buffer 1 and
keep the washed beads in a refrigerator until further use.
3.2 Starting Material
The preferred starting material is cryopreserved
human PBMC obtained from leukapheresis product
(5). The starting material can also be enriched for
specific T cell subsets, such as CD4+ T cells (8) or
CD25+ T cells (9, 10, 11).
For optimal T cell activation and expansion it is
This procedure is for separation (1, 2) and expansion (1, 5) of 5x108 CD3+ T cells. Adjust the volumes
accordingly when using higher/lower cell numbers.
1. Remove the required number of bags containing
cryopreserved apheresis cells from liquid N2
vapour storage.
3. To prevent cell clumping, add anti-coagulant solution aseptically to thawed cells to a final concentration of 10%.
4. Slowly dilute the cell suspension 1:1 in Buffer 1.
5. Wash the cells in Buffer 1 according to the cell
washer manufacturer’s recommendations.
6. Resuspend the cells in 50-60 ml of Buffer 1. If the
volume exceeds 60 ml, perform a centrifugation
step to reduce the volume.
7. Incubate the cells for 60 min in Buffer 1 at room
temperature to allow dead or dying cells to aggregate and subsequently be removed via a blood
filter as described below.
8. Filter the cells through an In-line Transfusion
Filter with cut-off between 40-80 µm. The cells
are now ready for further processing.
9. Remove 1 ml of the sample from the leukapheresis bag. Calculate the number of CD3+ T cells by
flow cytometry, and determine the viability of the
cells with trypan blue staining.
3
3.3.2 Magnetic Separation and Culture
of CD3+ T Cells
It has been shown that magnetic pre-selection of
CD3+ T cells can improve subsequent T cell expansion (1, 2).
The recommended standard procedure described
below uses a ratio of three Dynabeads ClinExVivo
CD3/CD28 per CD3+ T cell (Fig. 1).
[If the starting sample contains less than 25% CD3+
T cells, replace Buffer 1 with Incomplete Medium in
the procedures below.]
1. Dilute the cells to approx. 1 x 107 CD3+ T
cells/ml in Buffer 1. For the standard procedure
of processing 5 x 108 CD3+ T cells, dilute the
sample in 50-60 ml of Buffer 1.
T cell separation. Add
2. Use a 1L bag for
100 ml of air to the bag.
CD3+
3. Add 5 x 108 CD3+ T cells to the 1L bag in 50-60
ml Buffer 1.
4. Add 4.0 ml (corresponding to 1.6 x 109 Dynabeads) of washed Dynabeads ClinExVivo CD3/
CD28 per 5 x 108 CD3+ T cells and immediately
proceed to the next step.
5. Place the bag on a cell mixer and mix for 30 min
at room temperature to gently mix the cells and
the Dynabeads. [If the starting sample contains
less than 25% CD3+ T cells it may be beneficial
to mix the sample for 1-2 hours in Incomplete
Medium instead of Buffer 1. Optimise the mixing
temperature between 4-25°C for each application.]
6. Prepare 150 ml of Buffer 1 in a 300 ml bag.
7. After mixing, remove the 1L bag from the mixer
and drain 150 ml of Buffer 1 into the bag. [Note:
Handle the 1L bag very gently, to not disrupt the
bead/cell complexes.] Place the 1L bag directly
on the Dynal ClinExVivo MPC. Adjust the magnet
to a 60° angle.
8. Leave the bag on the Dynal ClinExVivo MPC for
1 min to capture the bead-bound CD3+ T cells.
While on the magnet, open the 1L bag containing the captured cells and drain waste fluid out
in waste bag via gravity. Remove the bag containing the captured cells from the magnet
immediately after all waste fluid has been drained.
9. Immediately add approximately 300 ml Complete Medium to the 1L bag containing the captured cells and gently resuspend the cell/bead
complexes.
10. Obtain a 3L bag
11. Transfer the cell/bead complexes from the 1L
bag into a 3L bag. Wash the 1L bag with Complete Medium and transfer the residual cells to
the 3L bag.
4
12. Repeat media wash of the 1L bag and transfer
the residual cells to the 3L bag until the volume
in the 3L bag has reached 1000 ml.
13. Place the 3L bag in an incubator at 37°C/ 5%
CO2 until Day 3 of culture. [Note; Using a bioreactor, e.g. Wave Bioreactor, will increase
expansion efficiency via perfusion and improved
aeration (rocking). Whereas typical cell densities rarely exceed 2-3 x 106 T cells/ml in static
cultures, bioreactor systems can readily maintain
viable T cells at densities of 2-4 x 107 T cells/ml.]
14. Collect a sample of the non-captured cell fraction
and manually count the number of non-captured
cells.
15. Stain the non-captured cells for CD3 expression
and evaluate by flow cytometry to calculate
depletion efficacy.
3.3.3 Counting and Splitting of Cultures
The cell concentration should be evaluated daily
beginning on day 3 of culture.
1. Gently mix the bag to help dissociate cell/bead
complexes and to resuspend the cells before removing 1-2 ml cell suspension for counting.
2. Again, mix the sample well to resuspend cells so
as to ensure maximum dissociation of beads from
cells. This will improve cell count accuracy.
3. Take an aliquot of cells and mix 1:1 with trypan
blue staining solution and manually count on a
hematocytometer (do not remove the Dynabeads
before counting).
Determine cell density and viability. [Caution:
insufficient mixing of bead/cell complexes may
result in cell count underestimates as they will
not migrate efficiently under hemocytometer
coverslips.]
4. Stain cells for CD3 expression and evaluate by
flow cytometry to calculate the number of CD3+
T cells in the bag.
5. Determine the total cell volume by weighing the
bag.
6. When CD3+ T cell density exceeds 1 x 106 cells/
ml, dilute the cells to approximately 0.5 x 106
CD3+ T cells/ml in Complete Medium.
7. Split the cultures to new 3L bags when needed.
[Note: T cell growth typically slows as T cell concentrations increase above 1-2 x 106 T cells/ml,
so adjust T cell numbers to ~0.5 x 106/ml to help
maintain the cells in log phase growth.]
8. Repeat counting of cells daily and dilute cells in
fresh Complete Medium to 0.5 x 106/ml.
3.3.4 Harvesting of Expanded CD3+ T Cells
1. Harvest cells on an optimal day for your application (usually day 8-12).
2. Remove the 3L bags from the incubator. Remove
a sample from a representative number of bags
for cell count and FACS analysis. Perform the cell
counts as described above.
per nucleated cell when monocyte levels are
>15% of total PBMC.
4. Place the bag into a humidified incubator at
37°C/ 5% CO2 for one hour.
3. Remove the beads by passing the cell culture
over the Dynal ClinExVivo MPC using gravitydriven flow. Determine the angle of the MPC
empirically between 0-60°. To achieve a flow rate
of up to 150 ml/min, the bags containing cells
and beads must be suspended from the pole so
that the fluid level is 85-90 cm above the cell collection bags.
[During the one hour incubation, beads and cells
come into contact via gravity at the bottom of the
bag and monocytes will ingest beads.]
5. Perform a final bead removal twice by placing the
bag on the Dynal ClinExVivo MPC. Adjust the
magnet to a 60° angle. After 1 min, drain the
cells into a new bag via gravity.
6. Allow ~50mL of Incomplete Medium to flow into
the bag containing the bead:cell complexes. Mix
well and transfer the medium containing residual
cells to the bag containing unbound cells.
4. Concentrate the cells and washed using a cell
washer.
6. Determination of residual beads can be performed as described in Reference 13.
3.3.5 Cryopreservation of Expanded CD3+ T
Cells
1. Prepare cryopreservation medium and cryopreserve the expanded T Cells.
2. Store the final product of expanded T cells in the
vapour phase of a liquid N2 storage unit.
3.4 Separation and Expansion of Fresh
(non-cryopreserved) Apheresis Cells
[Note: During thawing of cryopreserved PBMC, contaminating granulocytes tend to lyse, thereby diminishing inhibitory effects they may have upon T cell
expansion (5). If working with fresh, non-cryopreserved samples, allow for contaminating granulocytes.
It is recommended that for initiation of cultures with
non-cryopreserved samples, the Magnetic Concentration method described above is employed (See
3.3.2).
Overall T cell activation and expansion can be further improved by specifically depleting phagocytic
cells, such as monocytes, using Dynabeads
ClinExVivo Epoxy prior to magnetic concentration
and culture initiation (12, 13). This is particularly
useful when monocyte levels are >15% of total
PBMC.]
1. Dilute PBMC to 5-10 x 106 cells/ml in Incomplete
Medium (pre-warmed to 37°C).
2. Wash Dynabeads ClinExVivo Epoxy as described
above for Dynabeads ClinExVivo CD3/CD28 (see
3.1).
3. Add Dynabeads ClinExVivo Epoxy to the PBMC at
a 1-2 beads per total nucleated cells in a sterile
culture bag. It may be preferred to use 2 beads
5. Remove the bag of cells and beads from the
incubator after 1 hour. Gently mix the cells and
beads before removing Dynabeads ClinExVivo
Epoxy and any cells that have ingested beads
using a Dynal ClinExVivo MPC. Adjust the primary magnet to a 60° angle and transfer non-captured cells to a new 1L bag using gravity.
7. Count the cells in the bag, stain for CD3 and evaluate by flow cytometry to calculate the number
of CD3+ T cells in the sample.
8. Adjust the cell concentration to 1 x 107 CD3+ T
cells/ml in Incomplete Medium.
9. Perform capture of CD3+ T cells as described
above (Section 3.3.2). [Note: magnetic concentration should be done in Incomplete Medium in
place of Buffer 1 after monocyte/phagocytic cell
depletion using Dynabeads ClinExVivo Epoxy.]
10. Following capture of CD3+ T cells, prepare cells
for culture initiation as described in Section
3.3.2.
11. Place the bag with cells and Dynabeads
ClinExVivo CD3/CD28 in an incubator at 37°C/
5% CO2 until Day 3 of culture.
12. Count and split cell culture as described above
(Section 3.3.3).
3.5 Procedures Incorporating Gene
Transduction
Typically, for all culture conditions described earlier, T
cells from normal donor samples begin cycling and
start to divide between day 2 and 3 of culture (4, 8).
Day 1, 2 and/or 3 are recommended as optimal days
for transduction using lentivirus based vectors (14).
Magnetic removal of beads prior to transduction will
diminish overall cell expansion, but should not affect
viability. Leaving beads in during the retroviral transduction process should be acceptable for most transduction applications.
[Note: T cells obtained from patients with various
diseases and/or undergoing various treatments may
be slower at entering cell cycle and cell division may
not commence until 1, 2 or even 3 days later than
typically observed for samples from healthy donors.
For example, T cells from patients with HIV infection
5
may be slower to start cell cycling, as may be sammay
slower
to
cell
as
ples be
from
patients
undergoing
chemotherapy,
or
may
be
slower
to start
start
cell cycling,
cycling,
as may
may be
be samsamples
from
patients
chemotherapy,
or
patients
certainundergoing
kinds of cancer
(e.g. chronic
ples
fromwith
patients
undergoing
chemotherapy,
or
patients
with
certain
cancer
lymphocytic
(1, 8,of
Thus(e.g.
it is chronic
imporpatients
withleukemia)
certain kinds
kinds
of15).
cancer
(e.g.
chronic
lymphocytic
leukemia)
(1,
8,
15).
Thus
it
important
to
monitor
T
cell
activation
markers,
as
lymphocytic leukemia) (1, 8, 15). Thus it is
issuch
important
monitor
cell
activation
markers,
as
CD25,to
well asT
to determine
optimal
tant
toas
monitor
Tcell
celldivision
activation
markers, such
such
as
CD25,
well
division
optimal
splittingas
and
timingto
gene modificatiCD25,
asschedules
well as
as cell
cell
division
tofordetermine
determine
optimal
splitting
schedules
on.]
splitting
schedules and
and timing
timing for
for gene
gene modificatimodification.]
on.]
4. GENERAL INFORMATION
4.
GENERAL INFORMATION
4.
INFORMATION
4.1GENERAL
Certification
4.1
Certification
4.1
Certification
Invitrogen
Dynal AS conforms to the Quality
Invitrogen
Dynal
conforms
to
Quality
Systems
9001:2000
ISO
Invitrogen Standard
Dynal AS
ASISO
conforms
to the
theand
Quality
Systems
Standard
ISO
and
13485:2003
with the following
scope:
Systems
Standard
ISO 9001:2000
9001:2000
and ISO
ISO
13485:2003
with
the
13485:2003
with
the following
following scope:
scope:
"Development,
manufacturing,
marketing and sales
®, Dynabeads® marketing
"Development,
manufacturing,
and
and associated
proof
Dynospheres
"Development, ®
manufacturing,® marketing
and sales
sales
associated
proof
Dynospheres
ducts
to customers
that work
within
immunology,
®,, Dynabeads
® and
Dynabeads
and
associated
proof
Dynospheres
ducts
to
work
immunology,
biological
and clinicalthat
research,
cell based
therapy
ducts
to customers
customers
that
work within
within
immunology,
biological
and
clinical
diagnostics."
and in vitro
biological
and
clinical research,
research, cell
cell based
based therapy
therapy
in
diagnostics."
and
in vitro
vitro
diagnostics."
and
In the
United
States, Dynabeads ClinExVivo
ClinExVivo
In
the
States,
Dynabeads
CD3/CD28
is available
for use
in clinical trials
under
ClinExVivo
In
the United
United
States,
Dynabeads
CD3/CD28
is
available
for
an approved
or IDE.
CD3/CD28
is IND
available
for use
use in
in clinical
clinical trials
trials under
under
an
an approved
approved IND
IND or
or IDE.
IDE.
4.2 USA (Device Master File)
4.2
USA
Master
File)
4.2
USA (Device
(Device
Master
A Device
Master File
is heldFile)
with the United States
A
Device
Master
File
is
held
with
United
Food
&
Drug
Administration
which
willStates
assist
A Device Master File is held (FDA),
with the
the
United
States
Food
Drug
Administration
which
will
users &
application (FDA),
for FDA
approvals
on
Food
&with
Drugtheir
Administration
(FDA),
which
will assist
assist
users
with
application
for
on
their clinical
trials.
If cross-referencing
the Device
users
with their
their
application
for FDA
FDA approvals
approvals
on
their
clinical
If
the
Master
File istrials.
of interest
to an Investigational
New
their
clinical
trials.
If cross-referencing
cross-referencing
the Device
Device
Master
File
of
an
New
Drug (IND)
Application
orto
other
applications, please
Master
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is
of interest
interest
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(IND)
Application
or
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contact
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cation.
4.3 Technical Service
4.3
Technical
Service
4.3
Technical
Service Dynal AS for further techPlease
contact Invitrogen
Please
contact
Invitrogen
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nical information
at www.invitrogen.com
Please
contact Invitrogen
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AS for
for further
further techtechnical
information
at
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nical
information
at www.invitrogen.com
Certificate
of Analysis
(CoA) is available upon
Certificate
of
request.
Certificate
of Analysis
Analysis (CoA)
(CoA) is
is available
available upon
upon
request.
request.
4.4 Precautions
4.4
Precautions
4.4
Precautions
Material
Safety Data Sheet (MSDS) is available at
Material
http://www.invitrogen.com.
Material Safety
Safety Data
Data Sheet
Sheet (MSDS)
(MSDS) is
is available
available at
at
http://www.invitrogen.com.
http://www.invitrogen.com.
5. WARNINGS AND LIMITATIONS
5.
WARNINGS AND
LIMITATIONS
5.
LIMITATIONS
ForWARNINGS
research useAND
only/not
involving the generation
For
research
only/not
of
T
cells
for
use
in
therapy.
For research use only/not involving
involving the
the generation
generation
of
T cells
in
of
cells for
for use
usecells
in therapy.
therapy.
If Tmammalian
are used in this procedure,
If
mammalian
cells
used
this
appropriate
laboratory
guidelines
followed.
If
mammalian
cells are
are
used in
inmust
thisbeprocedure,
procedure,
appropriate
laboratory
guidelines
must
Detailed
information
on
such
guidelines
can be
appropriate laboratory guidelines must be
be followed.
followed.
Detailed
on
guidelines
can
obtained information
from: Protection
of Laboratory
Detailed
information
on such
such
guidelines Workers
can be
be
obtained
from:
Laboratory
from Instrument
Biohazardsof
and
Infectious Workers
Disease
obtained
from: Protection
Protection
of
Laboratory
Workers
from
from Instrument
Instrument Biohazards
Biohazards and
and Infectious
Infectious Disease
Disease
6
6
6
Transmitted by Blood, Body fluids and Tissue:
Transmitted
by
Body
fluids
and
Approved Guideline:
M29-A;
1-56238-339-6;
Transmitted
by Blood,
Blood,
BodyISBN
fluids
and Tissue:
Tissue:
Approved
Guideline:
http//:www.nccls.org.
Approved
Guideline: M29-A;
M29-A; ISBN
ISBN 1-56238-339-6;
1-56238-339-6;
http//:www.nccls.org.
http//:www.nccls.org.
Handle all samples as if capable of transmitting disHandle
samples
as
of
disease. Allall
should
be
performed
wearing gloves
Handle
allwork
samples
as if
if capable
capable
of transmitting
transmitting
disease.
All
be
and appropriate
protection.
ease.
All work
work should
should
be performed
performed wearing
wearing gloves
gloves
and
and appropriate
appropriate protection.
protection.
5.1 Trademarks and Patents
5.1
Trademarks
and
Patents
®, Dynabeads
®, ClinExVivo
5.1
Trademarks
and
Patents ™ and Dynal MPC™
Dynal
®, Dynabeads®, ClinExVivo™ and Dynal MPC™
Dynal
are
either
registered
trademarks
of
®
®
andtrademarks
Dynal MPC™
Dynal , Dynabeads , ClinExVivo™ or
are
either
trademarks
trademarks
of
Invitrogen
Dynal
AS, Oslo,
Norway.or
registration
are
either registered
registered
trademarks
orAny
trademarks
of
Invitrogen
Dynal
AS,
Any
or trademark
symbols
usedNorway.
herein denote
the regiInvitrogen
Dynal
AS, Oslo,
Oslo,
Norway.
Any registration
registration
or
trademark
used
denote
registration
statussymbols
of trademarks
in the
Unitedthe
States.
or
trademark
symbols
used herein
herein
denote
the
registration
status
of
trademarks
in
the
Trademarks
may
may not be
other
stration
status
of or
trademarks
in registered
the United
UnitedinStates.
States.
Trademarks
countries. may
Trademarks
may or
or may
may not
not be
be registered
registered in
in other
other
countries.
Ficoll® is a registered trademark of GE Healthcare
countries.
®
Ficoll
Bio-Sciences
® is
is aa registered
registered trademark
trademark of
of GE
GE Healthcare
Healthcare
Ficoll
Bio-Sciences
Bio-Sciences
Proleukin® is a registered trademark of Chiron
® is a registered trademark of Chiron
Proleukin
Corporation
® is a registered trademark of Chiron
Proleukin
Corporation
Corporation
X-vivo 15™ is a trademark of Cambrex.
X-vivo
15™
of Cambrex.
® isis
X-vivo
is aaa trademark
trademark
Cambrex. of Baxter
registeredoftrademark
Lifecell15™
® is a registered trademark of Baxter
Lifecell
International
Inc.
®
Lifecell is a registered trademark of Baxter
International
Inc.
®
®
International
is a registered trademark of Terumo
Terumo
TSCDInc.
TerumoTSCD
isaregisteredtrademarkofTerumo
® isJapan.
TM
aa registered
trademark
of
Terumo
Terumo
TSCD
KK
Corporation,
OpTmizer
T-cell
KK
Corporation,
®
registered
trademark
of ExspanTerumo
Terumo TSCD isJapan.
sionSFMisatrademarkofInvitrogenCorporation.
KK
KK Corporation,
Corporation, Japan.
Japan.
5.2 Intellectual Property Disclaimer
5.2
Intellectual
Property
Disclaimer
5.2
Intellectual
Property
Invitrogen
Dynal will
not be Disclaimer
responsible for violatiInvitrogen
Dynal
will
not
be
responsible
for
violations or patent
infringements
may occur
the
Invitrogen
Dynal
will not bethat
responsible
forwith
violations
or
patent
infringements
that
may
occur
with
use
of
our
products.
ons or patent infringements that may occur with the
the
use
use of
of our
our products.
products.
5.3 Warranty
5.3
Warranty
5.3
The Warranty
products are warranted to the original purchaThe
are
the
purchaser
only
to conform
to the to
quantity
and contents
The products
products
are warranted
warranted
to
the original
original
purchaser
only
conform
the
stated
onto
vial andto
labels forand
the contents
duration
ser
only
tothe
conform
toouter
the quantity
quantity
and
contents
stated
on
vial
labels
for
duration
of the stated
life. outer
Invitrogen
stated
on the
theshelf
vial and
and
outer
labelsDynal's
for the
theobligation
duration
of
stated
shelf
Invitrogen
Dynal's
obligation
andthe
purchaser's
this
of
thethe
stated
shelf life.
life.exclusive
Invitrogenremedy
Dynal'sunder
obligation
and
the
purchaser's
exclusive
remedy
under
warranty
is limited exclusive
either toremedy
replacement,
at
and
the purchaser's
under this
this
warranty
limited
either
at
Invitrogen is
Dynal's
expense,
products which
warranty
is
limited
either ofto
toanyreplacement,
replacement,
at
Invitrogen
Dynal's
expense,
of
products
shall be defective
manufacture,
whichwhich
shall
Invitrogen
Dynal's in
expense,
of any
any and
products
which
shall
be
in
and
be returned
to Invitrogen
Dynal, transportation
preshall
be defective
defective
in manufacture,
manufacture,
and which
which shall
shall
be
returned
to
Dynal,
transportation
paid,
or at Invitrogen
Dynal's
option,
refund ofprethe
be
returned
to Invitrogen
Invitrogen
Dynal,
transportation
prepaid,
or
Invitrogen
purchase
paid,
or at
atprice.
Invitrogen Dynal's
Dynal's option,
option, refund
refund of
of the
the
purchase
price.
purchase
Claims forprice.
merchandise damaged in transit must be
Claims
for
merchandise
submitted
to
the carrier.damaged
Claims for merchandise
damaged in
in transit
transit must
must be
be
submitted
to
carrier.
submitted
to the
the
carrier.
This warranty
shall
not apply to any products which
This
shall
not
to
products
which
shall warranty
have been
outside
Invitrogen
This
warranty
shallaltered
not apply
apply
to any
any
products Dynal,
which
shall
have
outside
nor shall
apply altered
to any products
which haveDynal,
been
shall
haveit been
been
altered
outside Invitrogen
Invitrogen
Dynal,
nor
shall
apply
to
which
have
been
subjected
misuse
or products
mishandling.
OTHER
nor
shall it
it to
apply
to any
any
products
whichALL
have
been
subjected
to
or
ALL
OTHER
WARRANTIES,
EXPRESSED,
IMPLIED OR
STATUTOsubjected
to misuse
misuse
or mishandling.
mishandling.
ALL
OTHER
WARRANTIES,
EXPRESSED,
IMPLIED
OR
RY, ARE HEREBY
SPECIFICALLY
EXCLUDED,
INCLUWARRANTIES,
EXPRESSED,
IMPLIED
OR STATUTOSTATUTORY,
ARE
HEREBY
SPECIFICALLY
EXCLUDED,
INCLUDING
BUT
NOT LIMITED
TO WARRANTIES
MERRY,
ARE
HEREBY
SPECIFICALLY
EXCLUDED,OF
INCLUDING
BUT
TO
OF
CHANTABILITY
OR FITNESS
FOR A PARTICULAR
DING
BUT NOT
NOT LIMITED
LIMITED
TO WARRANTIES
WARRANTIES
OF MERMERCHANTABILITY
OR
A
PURPOSE. Invitrogen
Dynal's FOR
maximum
liability is
CHANTABILITY
OR FITNESS
FITNESS
FOR
A PARTICULAR
PARTICULAR
PURPOSE.
Dynal's
liability
is
limited in allInvitrogen
events to the
pricemaximum
of the products
sold
PURPOSE.
Invitrogen
Dynal's
maximum
liability
is
limited
in
of
products
sold
by Invitrogen
Dynal.to
NOprice
EVENT
SHALL
INVITROlimited
in all
all events
events
toINthe
the
price
of the
the
products
sold
by
Invitrogen
Dynal.
IN
EVENT
INVITROGEN
DYNAL BE
LIABLE
FOR
ANYSHALL
SPECIAL,
INCIby
Invitrogen
Dynal.
IN NO
NO
EVENT
SHALL
INVITROGEN
GEN DYNAL
DYNAL BE
BE LIABLE
LIABLE FOR
FOR ANY
ANY SPECIAL,
SPECIAL, INCIINCI-
DENTAL OR CONSEQUENTIAL DAMAGES. Some states do not allow limits on warranties, or on remedies for breach in certain transactions. In such states,
the limits set forth above may not apply.
5.4 Limited Use Label License
No. 5: Invitrogen Technology - The purchase of this
product conveys to the buyer the non-transferable
right to use the purchased amount of the product
and components of the product in research conducted by the buyer (whether the buyer is an academic
or for-profit entity). The buyer cannot sell or otherwise transfer (a) this product (b) its components or
(c) materials made using this product or its components to a third party or otherwise use this product
or its components or materials made using this product or its components for Commercial Purposes.
The buyer may transfer information or materials
made through the use of this product to a scientific
collaborator, provided that such transfer is not for
any Commercial Purpose, and that such collaborator
agrees in writing (a) not to transfer such materials
to any third party, and (b) to use such transferred
materials and/or information solely for research and
not for Commercial Purposes. Commercial Purposes
means any activity by a party for consideration and
may include, but is not limited to: (1) use of the
product or its components in manufacturing; (2) use
of the product or its components to provide a service, information, or data; (3) use of the product or
its components for therapeutic, diagnostic or prophylactic purposes; or (4) resale of the product or its
components, whether or not such product or its
components are resold for use in research.
Invitrogen Corporation will not assert a claim
against the buyer of infringement of patents owned
or controlled by Invitrogen Corporation which cover
this product based upon the manufacture, use or
sale of a therapeutic, clinical diagnostic, vaccine or
prophylactic product developed in research by the
buyer in which this product or its components was
employed, provided that neither this product nor
any of its components was used in the manufacture
of such product. If the purchaser is not willing to
accept the limitations of this limited use statement,
Invitrogen is willing to accept return of the product
with a full refund.
No. 305: Invitrogen T Cell Expansion Technology The use of this product for ex vivo activation or
expansion of human T-cells is covered under various
patents owned and licensed from the U.S. Navy, the
University of Michigan and the Dana Farber Cancer
Institute and is sold for research use only. This product is not to be used for activation or expansion of
T-cells modified through gene transfer to specifically
modify the T-cells to produce secreted or cell-surface membrane-bound proteins not normally
expressed in significant levels by such T-cells, unless
the proteins directly enable the selection, or directly
modify or preserve the function, of the T-cells.
Modifications that are acceptable would include, for
example, introducing genes that direct expression of
cell-surface bound proteins that bind the T-cell to
specific target cells.
For information on purchasing a license to this product for purposes other than research, contact
Licensing Department, Invitrogen Corporation, 1600
Faraday Avenue, Carlsbad, California 92008.
Phone (760) 603-7200. Fax (760) 602-6500.
Email: [email protected]
6. REFERENCES
1. Bonyhadi M et al. (2005) In vitro engagement of
CD3 and CD28 corrects T cell defects in chronic
lymphocytic leukaemia. J Immunol. 174:23662375.
2. Parmar S et al. (2006) Ex vivo expanded umbilical cord blood T cells maintain naive phenotype
and TCR diversity. Cytotherapy 8:149-157.
3. Coito S et al. (2004) Retrovirus-mediated gene
transfer in human primary T lymphocytes induces an activation- and transduction/selectiondependent TCR-B variable chain repertoire skewing of gene-modified cells. Stem Cells and
Development 13:71-81.
4. Levine BL et al. (1997) Effects of CD28 costimulation on long-term proliferation of CD4+ T
cells in the absence of exogenous feeder cells. J
Immunol. 159: 5921-5930.
5. Hami L et al. (2003) Comparison of a static process and a biorector-based process for the GMP
manufacture of autologous Xcellerated T cells for
clinical trials. BioProcessing Journal 2:1-10.
6. Kalamasz D et al. (2004) Optimization of human
T-cell expansion ex vivo using magnetic beads
conjugated with ani-CD3 and anti-CD28 antibodies. J Immunother. 27:405-418.
7. Noonan K et al. (2005) Activated marrow-infiltrating lymphocytes effectively target plasma
cells and their clonogenic precursors. Cancer Res
65:2026-2034.
8. Levine BL et al. (1996) Antiviral effect and ex
vivo CD4+ T cell proliferation in HIV-positive
patients as a result of CD28 costimulation.
Science 272:1939-1943.
9. Earle KE et al. (2005) In vitro expanded human
CD4+CD25+ regulatory T cells suppress effector
T cell proliferation. Clinical Immunology 115:3-9.
10. Godfrey WR et al. (2004) In vitro-expanded
human CD4+CD25+ T-regulatory cells can markedly inhibit allogeneic dendritic cell-stimulated
MLR cultures. Blood 104:453-461.
7
11. Godfrey WR et al. (2005) Cord blood
CD4+CD25+-derived T regulatory cell lines
express FoxP3 protein and manifest potent
suppressor function. Blood 105:750-758.
12. Porter DL et al. (2006) A phase 1 trial of donor
lymphocyte infusions expanded and activated ex
vivo via CD3/CD28 costimulation. Blood
107:1325-1331.
13. Levine BL et al. (1998) Large-scale production of
CD4+ T cells from HIV-1-infected donors after
CD3/CD28 costimulation. J Hematother. 7:437448.
14. Bondanza A et al. (2006) Suicide gene therapy
of graft-versus-host disease induced by central
memory human T lymphocytes. Blood 107:
1828-1836.
15. Mitsuyasu RT et al. (2000) Prolonged survival
and tissue trafficking following adoptive transfer
of CD4ζ gene-modified autologous CD4+ and
CD8+ T cells in human immunodeficiency virusinfected subjects. Blood 96:785-793.
Invitrogen Dynal is a part of the Invitrogen Group.
Contact details for your local Invitrogen sales
office/technical support can be found at
http://www.invitrogen.com/contact
©Copyright2008InvitrogenDynalAS,Oslo,Norway.
©
Copyright 2007 Invitrogen Dynal AS, Oslo, Norway.
Allrightsreserved.
All
rights reserved.
SPEC-05371
Revised:
02.2007
Printed: 02.2007
8