GMP-MBS001.01 | 2023.12.18

GMP ActiveMax® Human T cell Activation/Expansion CD3/CD28 Beads

Cat. No. GMP-MBS001
Product Information

ProductSize

Amount10 mg

1×10 beads

100 mg(10 mg × 10)

1×109beads

400 mg(10 mg × 40)

4×109beads

8

GMP ActiveMax® Human T cell Activation/Expansion
CD3/CD28 Beads

ACROBiosystems provide rigorous quality control tests (fully validated equipment, processes and test methods) on our GMP grade
products to ensure that they meet stringent standards in terms of safety, activity and inter-batch stability, and each bulk QC lot
mainly contains the following specific information:
Test items



MethodSpecification

Physical appearance

Visual method

Brown liquid

Labelled quantity

Volumetric method

Not less than labelled quantity

Particle counting test

Counting using Cell counter5.00×107 beads/mL ± 10%

Antibody leakage assay

ELISA<0.50 μg/mL

T cell activation assay

FACS＞80% CD25/CD69 coexpression

T cell proliferation assay

CFSE stanning＞80% of proliferated T cells

Endotoxin

LAL method<0.5 EU/mL

Sterility <USP 71>

Membrane filtration method

NegativeFeatures
1. Designed in ISO 9001:2015 and ISO 13485:2016 certified facility
2. Manufactured and QC tested under a GMP compliance factory
3. Animal-Free materials
4. GMP grade antibodies as raw materials with strict virus removal steps and testing
5. GMP grade recombinant HSA as excipient
6. Batch-to-batch consistency
7. Stringent quality control tests

Product Description
GMP ActiveMax® Human T cell Activation/Expansion CD3/CD28 Beads are uniform 5.5 μm of magnetic beads coated with an
optimized mixture of GMP grade mouse monoclonal antibodies against the CD3 and CD28, mimicking in vivo stimulation by APCs.

1/8

GMP ActiveMax® Human T cell Activation/Expansion CD3/CD28 Beads are intended for the in vitro isolation, stimulation andexpansion of purified T cell populations of, for example, CD3+T cells, CD4+ T cells, CD8+ T cells or human PBMC for cell based
clinical and preclinical research.
GMP ActiveMax® Human T cell Activation/Expansion CD3/CD28 Beads are Manufactured under a GMP compliance factory withanimal free raw materials, and tested under GMP guidelines.

Formulation
GMP ActiveMax® Human T cell Activation/Expansion CD3/CD28 Beads is supplied with 5x107 beads/mL in PBS, pH 7.4, with 0.1%
recombinant human serum albumin (recombinant HSA).

Shipping and Storage
This product is supplied and shipped as sterile liquid solution with blue ice, please inquire the shipping cost.
The product MUST be stored at 2-8°C upon receipt;
This product is stable after storage at 2-8°C for 5 years under sterile conditions.

General guidelines
Because magnetic beads are 5.5 μm particle size, the beads may stick to the side of the bottle in the shipping process. Before
opening, it is recommended to gently shake the bottle to settle the beads. Avoid vigorous mixing such as vortex mixing. The beads
are dense and will tend to settle very quickly. Be sure that any bead mixture is homogenous before aliquoting.
Always work under sterile conditions to avoid contamination.

Prepare beads
Wash GMP ActiveMax® Human T cell Activation/Expansion CD3/CD28 Beads before use.
1. Resuspend the Magnetic Beads in the vial (i.e. vortex for >30 sec, or tilt and rotate for 5 min).
2. Transfer the desired volume of Magnetic Beads to a tube.
3. Add an equal volume of PBS buffer containing 1% HSA, or at least 1mL, and mix (vortex for 5 sec, or keep on a roller for at least 2min).
4. Place the tube on a magnet for 2 min and then discard the supernatant.
5. Remove the tube from the magnet and resuspend the washed Magnetic Beads in the same volume of T cell culture medium as
the initial volume of Magnetic Beads taken from the vial (step 2) .

Prepare cells
1. GMP ActiveMax® Human T cell Activation/Expansion CD3/CD28 Beads is intended for the in vitro stimulation and expansion ofpurified T cell populations of, for example, CD3+T cells, CD4+ T cells, CD8+ T cells or human PBMC.
2. Prepare T cell culture medium, such as RPMI1640 with 10% of FBS, CTS™ OpTmizer™ T-Cell Expansion SFM(Thermoscientific-gibco), X-VIVO-15 Serum-free Hematopoietic Cell Medium (Lonza) and other T cell culture medium.

Isolate human CD3+T Cells
The isolation protocol uses a beads-to-T cells ratio of 1:1, so must take some washed cells to calculate the viability,
concentration, and number of CD3+ T cells before starting cell isolation.
1. Take human PBMC from liquid nitrogen and thaw immediately at 37℃ for 5minutes.
2. Harvest the cells and wash once by PBS buffer containing 1% HSA，and count the cells number and the viability.
3. Detect the positive rate of CD3+T cells by flow cytometry，and calculate the number of CD3+T cells.
4. Adjust the cell density of 1 × 107 CD3+T cells with PBS buffer containing 1% HSA in 1.5mL or 4mL sterile centrifuge tube.
5. Add pre-washed and resuspended GMP ActiveMax® Human T cell Activation/Expansion CD3/CD28 Beads to obtain abeads-to-CD3+T cells ratio of 1:1 (The volume of cell-Beads suspension should not exceed half of the maximum volume of the2/8

GMP-MBS001.01 | 2023.12.18centrifuge tube).
6. Place the tube to 3D Rotating Mixer (MIULAB, RH-18+) and mix thoroughly for 30min at RT.
7. Transfer the tube on a magnet for 2 min to separate the beads-T cells from the solution.
8. Carefully pipette (do not pour) off the supernatant to a new tube, this is the unlabeled cell fraction.
9. Remove the tube from the magnet, this tube contains the isolated beads-cells complex.
10. Resuspend the isolated complex in the tube with the fresh T cell culture medium for downstream applications.
11. The supernatant collected from Step8 is used to calculate the isolated efficiency.

Note ： Calculation formula of isolated efficiency: The isolated efficiency of CD3+T cells=(1-the number of CD3+T cells in thenon-captured cells in step 8/the total number of CD3+T cells invested in step 4)*100%, Wherein, the number of CD3+T cells = thenumber of cells measured by cell counter * the positive rate of CD3+T cells)Activate Human T Cells
The following procedure describes activation of human T cells in 12-well tissue culture plate. Activation can also be performed inother cell culture devices, but the ratio of GMP ActiveMax® Human T cell Activation/Expansion CD3/CD28 Beads to T cells must be
1:1.
1. Start with 5× 106 purified T cells in 0.5 mL of T cell culture medium in a 12-well tissue culture plate.
2. Add 0.5 mL pre-washed and resuspended GMP ActiveMax® Human T cell Activation/Expansion CD3/CD28 Beads (2.5 × 106beads) to obtain a beads-to-cells ratio of 1:1.
3. Incubate in a humidified CO2 incubator at 37°C, according to your specific experimental requirements (Incubation time isrecommended for 24 hours).
4. Harvest the activated T cells and use directly for further analysis.
5. For flow cytometry applications, remove the beads prior to staining. Place the tube on a magnet for 2 min to separate the beads
from the solution. Transfer the supernatant containing the cells to a new tube.

Expand Human T Cells
1. Prepare T cell culture medium supplemented with 4ng/mL recombinant human IL-2 (rhIL-2).
2. Start with 1–1.5 × 106 purified T cells/mL in T cell culture medium in a suitable tissue culture plate or tissue culture flask.
3. Add GMP ActiveMax® Human T cell Activation/Expansion CD3/CD28 Beads at a beads-to-cells ratio of 1:1.
4. Incubate in a humidified CO2 incubator at 37°C, according to your specific experimental requirements.
5. Examine cultures daily, noting cell size and shape. Cell shrinking and reduced proliferation rate are typically observed inexhausted cell cultures.
6. When the cell density exceeds 2.0-2.5 × 106 cells/mL or when the medium turns yellow, split cultures back to a density of 4–8 ×
105 cells/mL with the complete T cell culture medium.

3/8

Typical Data

The human T cells were stimulated with GMP ActiveMax® Human T cell Activation/Expansion CD3/CD28 Beads (Cat. No. GMP-MBS001)
for 24hrs, and the activation was assessed by measuring expression of both activation markers CD25 and CD69 expression on the T cells
surface by stanning with PE labeled anti-human CD25 antibody and FITC labeled anti-human CD69 antibody respectively (QC tested).

The human T cells were labeled with carboxy fluorescein succinimidyl ester (CFSE) and stimulated with GMP ActiveMax® Human T cell
Activation/Expansion CD3/CD28 Beads (Cat. No. GMP-MBS001), and then the proliferation of the T cells was assessed with CFSE dilution
assay by flow Cytometry on day 5 after stimulation (QC tested).

4/8

Activation of the purified human T Cells. The purified human T cells were activated using Human T cell Activation/Expansion CD3/CD28Beads (ACRO, Cat. No. GMP-MBS001) and Competitor-Beads respectively for 24 hours with CTS Optimizer Medium. Cells werefluorescently stained using PE labeled anti-human CD25 antibody and labeled FITC anti-human CD69 antibody and analyzed by flowcytometry.

Expansion of the human CD3+T cells. Human T cells using ACROBiosystems CD3/CD28 Beads (ACRO, Cat. No. GMP-MBS001) wereexpanded under two different medium, respectively. Expansion was performed for two weeks, showing that ACROBiosystems’ beads
showing better proliferative abilities and comparable competitive ideas compared with competitor product.

5/8

PD-1 expression of the activated human T Cells. The purified human T cells were stimulated using Human T cell Activation/Expansion
CD3/CD28 Beads at a ratio of 1:1 beads-to-cells. Cells were expanded in T cell culture medium supplemented with 4ng/mL of rhIL-2
Protein. Activated T cells were expanded for up to 8 days with low PD-1 expression.

Stability

The Cell based assay shows that GMP ActiveMax® Human T cell Expansion CD3/CD28 Beads(Cat. No. GMP-MBS001) is stable at37℃ for 35 days.

Activation of the purified human T Cells. The purified human T cells were activated using Human T cell Activation/Expansion
CD3/CD28 Beads, (ACRO, Cat. No. GMP-MBS001/MBS-C001) respectively for 24 hours with CTS Optimizer Medium. Cells werefluorescently stained using PE labeled anti-human CD25 antibody and labeled FITC anti-human CD69 antibody and analyzed by flow
cytometry.

6/8

GMP-MBS001.01 | 2023.12.18

Expansion of the human CD3+T cells. Human T cells using ACROBiosystems CD3/CD28 Beads (ACRO, Cat. No.
GMP-MBS001/MBS-C001) were expanded under two different mediums, respectively. Expansion was performed for two weeks,showing that ACROBiosystems’ GMP and PG beads showed similar proliferative abilities.

PD-1 expression of the activated human T Cells. The purified human T cells were stimulated using Human T cell Activation/Expansion
CD3/CD28 Beads at a ratio of 1:1 beads-to-cells. Cells were expanded in T cell culture medium supplemented with 4ng/mL of rhIL-2
Protein. Activated T cells were expanded for up to 8 days with low PD-1 expression.

Contact Information
If you have any questions, please contact our technical support team at: TechSupport@acrobiosystems.com

MANUFACTURING SPECIFICATIONSACROBiosystems GMP grade products are produced under a quality management system and in compliance with relevant
guidelines: Ph. Eur General Chapter 5.2.12 Raw materials of biological origin for the production of cell-based and gene therapy
medicinal products; USP<92>Growth Factors and Cytokines Used in Cell Therapy Manufacturing; USP<1043>Ancillary Materials
for Cell, Gene, and Tissue-Engineered Products; ISO/TS 20399-1:2018, Biotechnology - Ancillary Materials Present During theProduction of Cellular Therapeutic Products.
ACROBiosystems Quality Management System Contents：
1. Designed in ISO 9001:2015 and ISO 13485:2016 certified facility, Manufactured and QC tested under a GMP compliance factory
2. Animal-Free materials
3. Materials purchased from the approved suppliers by QA
4. Qualified personnel
7/8

5. Quality-related documents review and approve by QA
6. Fully batch production and control records
7. Equipment maintenance and calibration
8. Validation of analytical procedures
9. Stability studies conducted
10. Comprehensive regulatory support files
Request For Regulatory Support Files（RSF）

8/8