An Ex Vivo Technique to Generate Tumor-Specific Chimeric Antigen Receptor T Cells

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Splenectomy and T Cell Preparation

Day 0:

1. Pour 10 ml of T cell media (TCM) into a 50 ml conical and place on ice for spleen collection.
2. Sacrifice the appropriate number of animals by CO₂ asphyxiation and secondary decapitation: Place animals in a cage receiving CO₂ at a flow rate of 10-30% cage volume/minute, per American Veterinary Medical Association guidelines (no more than 5 animals can be sacrificed simultaneously) until respiration terminates and for two minutes thereafter. Remove animals from the CO₂ chamber and decapitate.
   NOTE: One spleen of a 6–12-week-old female C57BL/6 mouse will yield approximately 4.5-5 x 10⁷ splenocytes. Here, 4 spleens will be harvested for approximately 200 x 10⁶ cells.
3. Lay the mouse such that its right side is facing up, and spray with 70% ethanol. With the forceps, grab a thin fold of skin below the left ribcage and cut a slight incision with the scissors. Peel back the skin, carefully grab a thin fold of the peritoneum with forceps, and cut a small cavity.
4. The spleen is a small, elongated, dark red organ that resembles a flattened bean; delicately grab the spleen with the forceps and excise by cutting away the surrounding connective tissue. Place the excised spleens into the conical containing 10 ml of TCM on ice.
5. Pour spleens over a 70 μm mesh cell strainer and disaggregate by mashing with the blunt end of the inside of a 5 ml syringe to generate a single-cell suspension. A maximum of two spleens should be disaggregated per mesh strainer.
6. Use a small volume of TCM to carefully wash the strainer following disaggregation to collect any remaining splenocytes. Pool all disaggregated spleens into a single-cell suspension. Bring final volume to 50 ml with TCM for one wash and spin at 300 x g for 10 min.
7. Prepare a solution of 1x lysis buffer by adding 5 ml 10x lysis buffer to 45 ml sterile water. To eliminate red blood cells, resuspend pellet in 5 ml of 1x lysis buffer per spleen in a 50 ml conical, mixing well by gently pipetting up and down. If exceeding 5 spleens, use a 250 ml centrifuge tube. Place conical or centrifuge tube in a 37 °C water bath for 5 min.
8. Remove the lysis reaction from the water bath and add TCM at a 1:1 ratio with lysis buffer to neutralize the reaction. Wash by spinning at 300 x g for 10 min.
9. Aspirate supernatant and fully resuspend pellet in TCM (2 ml/spleen, 8 ml total for 4 spleens) by pipetting up and down.
10. Count cells by adding 10 μl of cell suspension to 190 μl trypan blue (1:20 dilution). Multiply the number obtained in one of the four gridded squares by 20 x 10⁴ x total volume (8 ml) to obtain the total cell number (e.g., 125 cells x 20 x 10⁴ x 8 ml = 200 x 10⁶ splenocytes).
11. Dilute cells to a concentration of 2 x 10⁶ cells/ml in TCM, supplemented with 2 μg/ml concanavalin A (ConA) and 50 IU/ml recombinant human interleukin-2 (rhIL-2). Thus, for 200 x 10⁶ splenocytes, add 92 ml media to 8 ml cells, 200 μg ConA, and 5,000 IU rhIL-2.
12. Add 2 mL cells to each well of 24-well tissue-culture treated plates, such that 4 x 10⁶ cells are in each well (e.g., 100 ml of cells will require approximately 4 plates).
13. Incubate overnight at 37 °C with 5% CO₂.

2. Transduction

Day 1:

1. Calculate the number of non-tissue culture treated 24-well plates needed for transduction by multiplying the number of spleens harvested by 2 (8 plates are needed for 4 spleens).
2. Next, calculate the required volume of recombinant human fibronectin fragment (RHFF) solution needed to coat plates by multiplying (total number of wells + 3) x 0.5 ml (8 plates x 24 wells = 192 + 3 = 195) x 0.5 ml = 97.5 ml.
3. Prepare 97.5 ml phosphate-buffered saline (PBS) containing RHFF at a concentration of 25 μg/ml by multiplying the total volume by 25 μg (97.5 x 25 = 2437.5 μg). Add this amount of RHFF to 97.5 ml PBS.
4. Coat non-tissue culture treated 24-well plates by adding 0.5 ml PBS/RHFF solution per well.
5. Incubate overnight at 4 °C.

Day 2:

6. Dump PBS/RHFF solution from non-tissue culture treated 24-well plates.
7. Add 1 ml/well 2% bovine serum albumin (BSA) in PBS and incubate at room temperature for 30 min.
8. Remove BSA by firmly upending plate. Wash by adding 2 ml PBS.
9. Collect viral supernatant by transferring media from each Human embryonic kidney 293T (HEK293T)-poly-D-lysine (PDL) coated plate into a 250 ml centrifuge tube and spin 10 min at 500 x g.
10. Carefully transfer viral supernatant into a fresh 250 ml centrifuge tube, being sure not to disturb the cell pellet that may have formed.
11. Add fresh TCM to the viral supernatant such that the final volume is 3 ml more than the amount needed for RHFF-coated wells. For example, for 192 RHFF-coated wells, bring the volume of viral supernatant to 192 + 3 ml = 195 ml.
12. Remove cultured splenocytes from the incubator and resuspend by gently pipetting up and down 2 – 3 times in each well. Transfer to a 250 ml conical. If using a multichannel pipette, using a sterile reservoir prior to transfer will help expedite this step. Count as previously described and spin at 300 x g for 10 min.
13. Add recombinant human interleukin-2 (rhIL-2) to viral supernatant at a concentration of 50 IU/ml. For example, add 50 x 195 = 9,750 IU rhIL-2 to 195 ml of viral supernatant.
14. Resuspend splenocytes in viral supernatant at 1 x 10⁶ cells/ml
15. Add 1 ml/well of splenocyte suspension to RHFF-coated 24-well plates.
16. Spin for 90 min according to the following settings: 770 x g, acceleration = 4, brake/deceleration = 0, 32 °C.
17. Prepare a TCM solution with 50 IU/ml rhIL-2. For example, prepare a 200 TCM solution by adding 10,000 IU rhIL-2. Add 1 ml of rhIL-2/TCM to each well after centrifugation.
18. Culture overnight at 37 °C in 5% CO₂.

3. CAR T cell Culture and Harvest

Days 3 and 4:

1. If T cells achieve >80% confluence, cells may be split (this usually occurs by day 3 or day 4). To split cells, gently pipet up and down in each well 2-3 times, and move 1 ml from each well into new wells of a fresh 24-well tissue-culture treated plate. Then, add 1 ml of fresh TCM with 50 IU/ml IL-2 to each well such that final volume is 2 ml in all wells.
2. If cells do not reach >80% confluence, perform a half media change by slowly pipetting off 1 ml of media from the top of each well. Avoid disturbing cells settled on the bottom while removing media. Add 1 ml of fresh TCM containing 50 IU/ml rhIL-2.

Day 5:

3. Resuspend CAR T cells by gently pipetting up and down 3 times in each well and transfer to a 250 ml centrifuge tube. Spin cells at 300 x g for 10 min.
4. Completely aspirate supernatant without disturbing pellet. Wash once with PBS, count cells as previously described, and wash with PBS a second time. A typical CAR T cell yield is approximately 1 x 10⁶ cells per well (8 plates x 24 wells = 192 x 10⁶ CAR T cells).
5. Resuspend cells in PBS at a concentration of 5 x 10⁷/ml for an injection of 1 x 10⁷ in a volume of 200 μg (e.g., for 192 x 10⁶ CAR T cells, resuspend washed pellet in 3.84 ml PBS).