Dynabeads® CD3CD28 CTS
互联网
实验原理
实验试剂
10 ml of Dynabeads® CD3⁄CD28 CTS™
DynaMag™ CTS™ (Magnetic Particle concentrator)
Dynal® MPC™-1 (Magnetic Particle concentrator)
PBS (Gibco ) [Note: PBS must be calcium and magnesium free.]
Anti-coagulant solution e.g. ACD-A, Baxter)
实验设备
1-Bags (Baxter Lifecell® or CellGenix Vuelife™)
3-L Culture Bags (Baxter Lifecell® or CellGenix Vuelife™)
Sampling site coupler with female luer (Charter Medical)
Terumo TSCD® Sterile Tubing Welder
40μm - 80μm In-line Transfusion Filter (Pall)
10-lead harness sets (compatible with Terumo SCD 312 welder), (Charter Medical)
COBE 2991 Cell Washer Disposable Set (COBE/Gambro) or Cytomate Disposable Set (Baxter)
Cell mixer (e.g. Heidolph Polymax 2040)
Plasma thawing device (e.g. Thermogenesis MT202)
Bioreactor (e.g. Wave Bioreactor)
实验步骤
1. Dynabeads® Washing Protocol
2) Place one 10 ml vial of Dynabeads® CD3⁄CD28 CTS™ on the MPC-1 for 1 min.
3) Aseptically remove the supernatant.
4) Remove the vial from the MPC-1.
5) Aseptically add 10 ml Buffer 1 to the vial and mix.
6) Place the vial on the MPC-1 for 1 min.
7) Aseptically remove the supernatant.
2. Thawing and Washing of Cryopreserved Apheresis Cells
2) Thaw the bag(s) in a plasma thawing device or by equivalent methods.
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.
3. Magnetic Separation and Culture of CD3 T Cells
2) Use a 1L bag for CD3 T cell separation. Add 100 ml of air to the bag.
3) Add 5 x 108 CD3 T cells to the 1L bag in 50-60 ml Buffer 1.
6) Prepare 150 ml of Buffer 1 in a 300 ml bag.
4. Counting and Splitting of Cultures
The cell concentration should be evaluated daily beginning on day 3 of culture.
5) Determine the total cell volume by weighing the bag.
8) Repeat counting of cells daily and dilute cells in fresh Complete Medium to 0.5 x 106 /ml.
5. Harvesting of Expanded CD3 T Cells
1) Harvest cells on an optimal day for your application (usually day 8-12).
4) Concentrate the cells and washed using a cell washer.
6) Determination of residual beads can be performed.
6. 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.
7. Separation and Expansion of Fresh (non-cryopreserved) Apheresis Cells
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® CD3⁄CD28 CTS™.
8) Adjust the cell concentration to 1 x 107 CD3 T cells/ml in Incomplete Medium.
10) Following capture of CD3 T cells, prepare cells for culture initiation.