An Ex Vivo Technique to Manufacture Chimeric Antigen Receptor T Cells

Protokol

All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines.

1. T Cell Activation, Transduction, and Expansion

1. Activate fresh or cryopreserved primary human T cells by mixing with anti-CD3/CD28 magnetic beads (e.g., dynabeads) at a ratio of 3 beads per T cell in 6-well cell culture dishes. Culture T cells in X-VIVO 15 medium supplemented with 5% normal human AB serum, 2 mM L-glutamine, 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), and interleukin 2 (IL2, 100 units/mL). Maintain T cells at a concentration of 10⁶ T cells/mL during expansion. Culture T cells at 37 °C, 20% O₂, and 95% humidity with 5% CO₂.
2. After overnight stimulation, add lentiviral supernatant to activated T cells. Calculate the volume of supernatant necessary to achieve a multiplicity of infection (MOI) of 3−5.
   NOTE: The CD19-BBζ CAR lentivirus plasmid consists of a CD8 hinge, 4-1BB costimulatory domain, and CD3ζ signaling domain. CD19-BBζ lentiviral supernatant was generated as previously described.
3. On day 3, collect a representative aliquot of cells for cryopreservation. Prior to cryopreservation, remove the magnetic beads by gentle pipetting and magnetic separation. Prepare a freezing medium containing phosphate-buffered saline (PBS) with 0.5% dimethyl sulfoxide (DMSO) and store at 4 °C until use.
   1. Centrifuge T cells at 300 x g for 5 min. Discard the supernatant and add 5 mL of PBS. Centrifuge cells at 300 x g for 5 min and discard the PBS.
   2. Resuspend the cell pellet in 1 mL of cold cryopreservation medium. Freeze T cells in a chilled freezing container and store at -80 °C for 48 h. Transfer the frozen cells to liquid nitrogen.
4. Wash the rest of the T cells once in 5 mL of PBS to eliminate the residual vector. Centrifuge at 300 x g for 5 min. Decant the PBS and resuspend the cell pellet in a T cell culture medium at a concentration of 0.5 x 10⁶ cells/mL.
5. Split the T cells into two cultures, designated for day 5 and day 9. Count T cells by flow cytometry using counting beads (Table of Materials) and monoclonal antibodies to human CD4 and CD8, as well as a viability dye (Table of Materials).
   1. To measure T cell concentration, prepare a master mix containing 500 µL of PBS, 5 µL of counting beads, 10 µL of 7-Amino-actinomycin D cell viability solution, 4 µL of CD4-fluorescein isothiocyanate (FITC) and 4 µL of CD8-allophycocyanin (APC). Add 40 µL of T cells to the master mix and measure cell concentration by flow cytometry based on the number of live T cells/bead counts. Refeed to maintain the cultures at a concentration of 0.5 x 10⁶ cells/mL every other day.
6. On day 5, count and cryopreserve day 5 cultures as described in steps 1.3 and 1.5.
7. On day 7, wash 0.5 x 10⁶ T cells in PBS and resuspend in 100 µL of fluorescence-activated cell sorting (FACS) buffer. Detect CAR surface protein expression by immunostaining with a fluorescently-conjugated anti-CAR19 idiotype by flow cytometry.
8. On day 9, count day 9 cultures and cryopreserve as described in step 1.3.

Malzemeler

Name	Company	Catalog Number	Comments
Anti CD3/CD28 dynabeads	Thermo Fisher	40203D
APC Mouse Anti-Human CD8	BD Biosciences	555369	RRID:AB_398595
APC-H7 Mouse anti-Human CD8 Antibody	BD Biosciences	560179	RRID:AB_1645481
CountBright Absolute Counting Beads,	Invitrogen	C36950
HEPES	Gibco	15630-080
Human AB serum	Valley Biomedical	HP1022
Human IL-2 IS, premium grade	Miltenyi	130-097-744
L-glutamine	Gibco	28030-081
Multisizer Coulter Counter	Beckman Coulter
X-VIVO 15	Gibco	04-418Q