| Day 1 Activation |
1.Resuspend cells at 1×106/ml in multiple 100 ml Miltenyi bags; 2.Coat 2× number of T cell bags with retronectin (1 mg/ml in 10 ml PBS) |
| Day 3 Transduction Round 1 |
1.Thaw vector; 2.Remove RN from bags and add 50 ml vector per bag; 3.Spin bags at 1000 g, 40 min; 4.Transfer cell suspension to each bag (1∶1 ratio) |
| Day 4 Transduction Round 2 |
1.Thaw vector; 2. Remove RN from bags and add vector; 3. Spin bags at 1000 g, 40 min; 4. Volume reduce; 5. Add IL2 to final concentration 100 u/ml |
| Day 6 Culture |
Add IL2 to final concentration 100 u/ml |
| Day 7 Bead removal |
1.Assess CD34 expression by flow cytometry; 2 Remove CD3/CD28 beads using MagSep (Dynal); 3.Rest overnight in X-Vivo 10+5% AB serum+IL2 100 u/ml |
| Day 8 Positive selection |
1.CliniMacs selection of CD34+ T cells; 2.Rest overnight in X-Vivo 10+5% AB serum+IL2 100 u/ml |
| Day 9 Dose preparation |
1.Flow cytometry for CD34 purity; 2.Phenotype analysis by flow cytomtetry; 3.Archive samples for RCR testing; 4.Cryopreserve cells in dose aliquots |
