一、實驗原理

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.

二、實驗試劑

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).

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

rIL-2

三、實驗步驟

1. 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.

2. Isolation of Human Treg Cells

For isolation of human Treg cells it is recommended to use the Dynal CD4 CD25 Treg Kit.

3. 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.

4. 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.

（1）Before re-stimulation:

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

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

（2）Day 0:

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

ii. Add 50 μl Dynabeads Treg Expander.

iii. Add 100 U/ml rIL-2.

（3）Day 1:

i. Add 100 μl Culture Medium containing100 U/ml rIL-2 per well.

（4）Day 2 or 3:

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

（5） Day 5-7:

i. Split the culture when needed.

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

iii. 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.

（6）Day 8:

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

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

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

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

四、注意事項

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.