2-layer stack

132M PBMC + 300ng/ml Gm/Flt3L

in 26.5 ml XVIVO15

Bag 3: add 132.5 ml

contains 3x HS, 3x Gm/Flt3L

Gas perm bag

37M PBMC + 300ng/ml Gm/Flt3L + 1ug/ml peptide

in 18.5 ml XVIVO15

transfer all 18.5 ml + 2x 12/7/15/21 + 2x HS

in 18.5 ml XVIVO

37 ml total

Bag 1 = 15.72 ml

Bag 2 = 2.78 ml

add 18.5 ml total to make 3x HS

Bag 1 = 14.8 ml

Bag 2 = 3.7 ml

add 18.5 ml total to make 4x HS

Bag 1 = 10 ml

Bag 2 = 26.5 ml

add 132.5 ml total to make 4x HS

remove media + NA cells = W

add Bag 4: contains 1ug/ml peptide, add 260 ml

remove media = W

wash with Bag 1 260 ml

remove media = W

transfer 75 ml from T-175 into 2-layer stack

wash T-175 with Bag 1 = 55 ml and transfer to 2-layer stack

add Bag 5: 130 ml contains 2x HS, 2x high dose 12/7/15/21

Bag 1 = 110.5 ml

Bag 2 = 19.5 ml

add 130 ml total to make 3x HS

Harvest from 2-layer stack