An Ex Vivo Technique to Generate Tumor-Specific Chimeric Antigen Receptor T Cells
Transcript
On the day after the splenectomy, prepare PBS containing 25 micrograms per milliliter recombinant human Fibronectin fragment or RHFF and coat non-tissue culture-treated 24-well plates by adding 0.5 milliliters of the solution to each well. The following day, remove the solution from the wells, and add 1 milliliter per well of 2% BSA in PBS.
Incubate at room temperature for 30 minutes, then, remove the BSA by firmly upending the plate before adding 2 milliliters of PBS to wash. After collecting the viral supernatant from the HEK293T cells, add fresh TCM to a final volume in milliliters equal to the number of RHFF-coated wells plus an additional 3 milliliters.
Remove the cultured splenocytes from the incubator, and resuspend by gently pipetting up and down two to three times in each well. Transfer the cell suspensions to a 50-milliliter conical tube. Next, resuspend the splenocytes in viral supernatant to 1 x 106 cells per milliliter.
Then, add 1 milliliter per well of the splenocytes suspension to the wells of the RHFF-coated plates. Now, spin the plates for 90 minutes at 32 degrees Celsius and 770 gs with an acceleration of 4 and a break setting of zero. During the spin, prepare 50 international units per milliliter of recombinant human IL-2 in TCM.
After the spin, add 1 milliliter of the medium to each well of the plates and incubate overnight. If T cells reach greater than 80% confluence, split the cells by gently pipetting up and down in each well, two to three times, then transfer 1 milliliter from each well into new wells of a fresh 24-well tissue culture-treated plate.
Add 1 milliliter of fresh TCM, with 50 international units per milliliter of IL-2 to each well. If the cells do not reach greater than 80% confluence, change half the medium by slowly pipetting off 1 milliliter of medium from the top of each well and replacing it with 1 milliliter of fresh TCM, containing 50 international units per milliliter of recombinant human IL-2.
Resuspend CAR T cells by gently pipetting up and down three times in each well, and transfer to a 250-milliliter centrifuge tube. Spin the cells at 300 gs for 10 minutes. Next completely aspirate the supernatant without disturbing the pellet. Use PBS to wash the cells, then count them before washing a second time. A typical CAR T cell yield is approximately 1 x 106 cells per well.
