Introduction

This protocol is intended for activation and expansion of human Treg cells isolated with the Dynal® CD4+CD25+ Treg Kit (Cat. no. 113.23D). The expanded Treg cells retain their regulatory capacity (1-3).

Principle of expansion

Dynabeads® Human Treg Expander offers a simple method for expansion of Treg cells that does not require antigen- presenting cells or antigen. Just add Dynabeads® Human Treg Expander and recombinant IL-2 (rIL-2) to the Treg culture to expand the Treg cells. Cell cultures showing signs of exhaustion can be re-stimulated by adding fresh beads and rIL-2.

Ready-to-use Dynabeads® Human Treg Expander offer the first artificial antigen- presenting cells to provide simultaneous signals to TCR/CD3 and CD28 for full activation and expansion of human Treg cells.

Description of Materials

Dynabeads® Human Treg Expander are uniform 4.5 μm, superparamagnetic polystyrene beads coated with an optimized mixture of monoclonal antibodies against the CD3 and CD28 cell surface molecules of human T cells. The CD3 antibody coated on Dynabeads® Human Treg Expander is specific for the epsilon chain of human CD3, a subunit of the TCR complex. The CD28 antibody is specific for the human CD28 co-stimulatory molecule, which is the receptor for CD80 (B7-1) and CD86 (B7-2). Both antibodies are coupled to the same bead, mimicking In vivo stimulation by antigen presenting cells.

Materials Supplied

  • 2 ml Dynabeads® Human Treg Expander
  • 2 x 107 beads/ml in phosphate buffered saline (PBS), pH 7.4, w/ 0.1% human serum albumin (HSA).

Additional materials required

Materials that are not included, but are needed to perform the entire protocol:

  • Magnet: Any Dynal® MPC™
  • Buffer 1: PBS w/ 0.1% BSA, pH 7.4
  • Cultural Medium: OpTmizer™ T-Cell Expansion SFM (Gibco) serum free 1X formulation designed to support the culture and expansion of human T cells(or equivalent)
  • Round bottom tissue culture plates or tissue culture flasks of suitable size
  • Humidified CO2 incubator
  • Dynal® CD4+CD25+ Treg Kit (Cat. no. 113.23D)
  • rIL-2
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Ordering Information

Sku Name Size Price Qty
11363D Dynabeads® Regulatory CD4+/CD25+ T Cell Kit 2 x 5 mL USD 876.00

Protocol

Technical Advice

It is generally recommended to use a bead-to-cell ratio of 4:1 and the cell concentrations given in Table 1. Other bead-to-cell ratios and cell concentrations can be used following optimization for your particular application. At least 95% pure Treg cells will give the most optimal expansion results. Low purity will allow CD8+ and CD4+CD25- T cells to grow and reduces the number of Treg cells in relative terms.

If the purity is lower than recommended above, the expansion results might be sub-optimal. To improve the expansion results, the amount of IL-2 can be increased up to 1000 U/ml rIL-2.

Dynabeads® Washing Procedure

Dynabeads® should be washed before use with the aid of a magnet.

  1.    Resuspend the Dynabeads® in the vial.

  2.    Transfer the desired volume of Dynabeads® to a tube.

  3.    Add the same volume of Buffer 1 as the initial volume of Dynabeads®, or at least 1 ml, and mix.

  4.    Place the tube in a magnet for 1-2 minutes until the Dynabeads® are separated; discard the supernatant. Remove the tube from the magnet.

  5.    Resuspend the washed Dynabeads® in the same volume of Buffer 1 as the initial volume of Dynabeads®.

Table 1

Volume/Number of Treg Cells 1 X 105 Treg Cells 1 X 106 Treg Cells
Type of CulturePer well in 96-well tissue culture platePer well in 24-well tissue culture plate
Dynabeads® Treg Expander20 μl200 μl
rIL-2500 U/ml rIL-2500 U/ml rIL-2
Seeding Volume (medium)100 μl1 ml

Isolation of Human Treg Cells

For isolation of human Treg cells it is recommended to use the Dynal® CD4+CD25+ Treg Kit (Cat no. 113.23D).

Expansion of Human Treg Cells Day 0:

Start with 1 x 105 Treg cells in 100 μl Culture Medium in a round bottom 96 well tissue culture plate.
Add 20 μl Dynabeads® Human Treg Expander.
Add 500 U/ml rIL-2.

Day 1:

Add 100 μl Culture Medium containing
500 U/ml rIL-2.

Day 3:

Resuspend and split the culture in half and add 100 μl Culture Medium containing
500 U/ml rIL-2 per well.

Day 5-7:

Split the culture when needed. Examine cultures daily, noting cell size and shape. Cell shrinking and reduced proliferation rate is typically observed in exhausted cell cultures. Count the cells at least twice weekly after thorough resuspension.

When the cell density exceeds 2 x 106 cells/ml or when the medium becomes yellow, split cultures to a density of 0.5 - 1.0 x 106 cells/ml in Culture Medium containing 500 U/ml rIL-2. Grow the cells until the well is half-full (~500,000 cells) and transfer the cells from a 96 well to a 24 well plate.

Day 8:

Remove the Dynabeads® by resuspending the cells and transferring the cells to a suitable tube. Place the tube in a magnet for 1-2 minutes until the Dynabeads® are separated. Centrifuge the supernatant and resuspend the cell pellet in fresh Culture Medium containing 100 U/ml rIL-2. Split the cultures when needed and rest the cells until Day 21-24 in culture medium with 100 U/ml rIL-2. Incubate in a humidified CO2 incubator at 37°C.

Re-stimulation of Human Treg Cells

The cells can be re-stimulated 15-18 days after the first stimulation, or when cell shrinking and reduced rate of proliferation is observed. Guidelines for restimulation are provided in Table 2.

Before re-stimulation:

  1.    Count the cells and split the cultures to a density of 1 x 106 cells/ml in Culture Medium.

  2.    Use the volumes given in Table 2 and follow the protocol below.

Day 0:

  1.    Start with 1 x 106 Treg cells in 1 ml Culture Medium in a 24 well tissue culture plate.

  2.   Add 50 μl Dynabeads® Treg Expander.

  3.   Add 100 U/ml rIL-2.

Day 1:

  1.    Add 100 μl Culture Medium containing

  2.   100 U/ml rIL-2 per well.

Day 2 or 3:

  1.   Resuspend and split the culture in half and add 100 μl Culture Medium containing 100 U/ml rIL-2 per well.

Day 5-7:

  1. Split the culture when needed.

  2. Examine cultures daily, noting cell size and shape. Cell shrinking and reduced proliferation rate is typically  observed in exhausted cell cultures.

  3. Count the cells at least twice weekly after thorough resuspension.
When the cell density exceeds 2 x 106 cells/ml or when the medium becomes yellow, split cultures to a density of 0.5 - 1.0 x 106 cells/ml in Culture Medium containing 100 U/ml rIL-2.

Day 8:

  1.    Remove the Dynabeads® by resuspending the cells and transferring the cells to a suitable tube.

  2.    Place the tube in a magnet for 1-2 minutes until the Dynabeads® are separated.

  3.   Centrifuge the supernatant and resuspend the cell pellet in fresh Culture Medium containing 100 U/ml rIL-2.

  4.   Split the cultures when needed and rest the cells until Day 21-24 in culture medium with 100 U/ml rIL-2.

Table 2

Volume/Number of Treg Cells 1 X 105 Treg Cells 1 X 106 Treg Cells
Type of CulturePer well in 96-well tissue culture platePer well in 24-well tissue culture plate
Dynabeads® Treg Expander5 μl50 μl
rIL-2100 U/ml rIL-2100 U/ml rIL-2
Seeding Volume (medium)100 μl1 ml


Mixed Lymphocyte Reaction (MLR) Assay for Identification of Suppressive Capacity of Expanded Treg Cells

  1. Isolate dendritic cells and culture with TNF-a and LPS for 2 days and irradiate before use as stimulators in MLR.

  2. Establish co-cultures of 5 x 104 responder CD4+CD25- T cells and 1 x 104 irradiated allogenic dendritic cells as stimulators in 96-well U-bottom plates.

  3. Add freshly purified or in vitro expanded CD4+CD25+ Treg cells in ratios of 1:1, 1:4, 1:8, 1:16 and 1:32 (CD25+: CD25- ratio).

  4. Pulse the wells on day 6 with 3H-thymidine, and culture for an additional 18 hours before harvest
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General Information

Invitrogen Dynal® AS complies with the Quality System Standards ISO 9001:2000 and ISO 13485:2003.

Storage/Stability

This product is stable until the expiry date stated on the label when stored unopened at 2-8°C.
Store opened vials at 2-8°C and avoid bacterial contamination

Keep Dynabeads® in liquid suspension during storage and all handling steps, as drying will result in reduced performance. Resuspend well before use.

Technical Support

Please contact Invitrogen Dynal® AS for further technical support (see contact details).

Warnings and Limitations

This product is for research use only. Not intended for any animal or human therapeutic or diagnostic use unless otherwise stated. Follow appropriate laboratory guidelines.

This product contains 0.02% sodium azide as a preservative, which is cytotoxic. Avoid pipetting by mouth!
Sodium azide may react with lead and copper plumbing to form highly explosive metal azides. When disposing through plumbing drains, flush with large volumes of water to prevent azide build up.

Certificate of Analysis (CoA) is available upon request. Material Safety Data Sheet (MSDS) is available at .

Patents and Trademarks

Dynal®, Dynabeads® and Dynal MPC™ are either registered trademarks or trademarks of Invitrogen Dynal® AS, Oslo, Norway. Any registration or trademark symbols used herein denote the registration status of trademarks in the United States. Trademarks may or may not be registered in other countries.

Intellectual Property Disclaimer


Invitrogen Dynal® will not be responsible for violations or patent infringements that may occur with the use of our products.
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References

  1. Godfrey WR et al. (2004) In vitroexpanded human CD4+CD25+ T-regulatory cells can markedly inhibit allogeneic dendritic cell-stimulated MLR cultures. Blood 104(2): 453-461.

  2. Godfrey WR et al. (2005) Cord blood CD4+CD25+-derived T regulatory cell lines express FoxP3 protein and manifest potent suppressor function. Blood 105(2): 750-758.

  3. Hoffmann P et al. (2004) Large-scale in vitro expansion of polyclonal human CD4+CD25 high regulatory T cells. Blood 104(3): 895-903.
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111.29D.indd    Rev 003      5-May-2008