An Ex Vivo Technique to Manufacture Chimeric Antigen Receptor T Cells

Activate fresh or cryopreserved primary human T cells by mixing them with anti-CD3/CD28 magnetic beads at a ratio of 3 beads per T cell in the wells of the culture dishes. Culture the T cells in X-VIVO 15 medium supplemented with normal human AB serum, L-glutamine, HEPES, and IL-2.

Activate the T cells at a concentration of 1 million T cells per milliliter during expansion, and culture them at 37 degrees Celsius with 20% oxygen, 5% carbon dioxide, and 95% humidity. After overnight stimulation, add lentiviral supernatant to the activated T cells. Calculate the volume of supernatant necessary to achieve a multiplicity of infection between 3 and 5. Continue culturing the cells using the same conditions.

On day 3, collect a representative aliquot of the cells of cryopreservation. Prior to cryopreservation, remove the magnetic beads by gently pipetting and magnetic separation. Prepare the freezing medium which contains PBS with 0.5% DMSO and store it at 4 degrees Celsius until ready to use.

Next, centrifuge the T cells at 300 times g for 5 minutes. Discard the supernatant and add 5 milliliters of PBS. Centrifuge the cells again at 300 times g for 5 minutes and discard the PBS. Resuspend the pellet in 1 milliliter of cold cryopreservation medium. Freeze the T cells in a chilled freezing container and store at minus 80 degrees Celsius for 48 hours.

After this, transfer the frozen cells to liquid nitrogen. Wash the rest of the T cells once in 5 milliliters of PBS to eliminate any residual vector. Centrifuge at 300 times g for 5 minutes, then decant the PBS and resuspend the cell pellet in T cell culture medium at a concentration of 500,000 cells per milliliter.

Split the T cells into two cultures designed for day 5 and 9. Count the T cells by flow cytometry using counting beads and monoclonal antibodies to human CD4 and CD8 as well as a viability dye. Refeed the cultures every other day to maintain them at a concentration of 500,000 cells per milliliter.

On day 5, counter and cryopreserve the day 5 cultures as previously described. On day 7, wash 500,000 cells in PBS and resuspend them in 100 microliters of fluorescence-activated cell sorting buffer. Then detect CAR surface protein expression by immunostaining with a fluorescently-conjugated anti-CAR19 idiotype by flow cytometry. On day 9, count and cryopreserved the day 9 cultures as previously described.