Isolation, Cultivation, and Transient Transfection of Primary Human T Cells to Generate Chimeric Antigen Receptor (CAR) T Cells

My lab focuses on next-generation cell therapies with T-cells expressing chimeric antigen receptor, or short, CARs. T-cell lines only poorly represent a diversity of primary human T-cells. This protocol provides guidelines for isolating, engineering, and analyzing primary T-cells for the generation of CAR T-cells.

To begin, obtain primary T-cells at the exponential growth phase and dilute them to a density of 0.3 times 10 to the power six cells per milliliter in full T-cell medium one day before the experiment. On the day of transfection, pre-warm all required media at 37 degrees Celsius. Calculate the required seeding volume to achieve a final density of 0.5 to 1.0 times 10 to the power of six cells per milliliter after electroporation.

Add full T-cell medium in a 12-well plate to reach the required density and pre-warm the plate to 37 degrees Celsius until electroporation. Count the T-cells and calculate the required volume to use one to five times 10 to the power of six cells for each chimeric antigen receptor messenger ribonucleic acid delivery. Transfer the required volume of T-cell suspension into a conical centrifuge tube.

Spin the tube at 300g at room temperature for five minutes and discard the supernatant. Perform the first washing step by adding 20 milliliters of RPMI medium with phenol red and without supplements on top of the cell pellet. Centrifuge at 300g at room temperature for five minutes, discard the supernatant, and repeat the washing twice:once with RPMI without phenol red and supplements and once with reduced serum medium.

After the final wash, discard the supernatant thoroughly by pipetting off the remaining liquid without disturbing the pellet. Resuspend the cell pellet in 100 microliters of reduced serum medium per electroporation. During the final T-cell washing step, collect all required mRNA aliquots from the freezer and keep them on ice.

Label the electroporation cuvettes and the plate with the pre-warmed media and set up the conditions on the electroporation device using a Gene Pulser Xcell Total System. Select the Square Wave Protocol and set the required parameters. Once the T-cell suspension is ready, combine 100 microliters of the suspension with three to five micrograms of mRNA per electroporation and mix gently without pipetting up and down.

Now, transfer the mRNA and T-cell mixture to an electroporation cuvette. Ensure that the suspension fully covers the bottom of the cuvette without pipetting up and down. Next, quickly transfer the cuvette to the electroporation device and perform electroporation using the previously prepared settings.

After electroporation, add 100 microliters of pre-warmed full T-cell medium from the well plate to the cuvette to recover the T-cells. Transfer the cells back to the plate. Rinse the cuvette with an additional 100 microliters of medium and transfer the rinse to the same well.

Incubate the Transfected T-cells at 37 degrees Celsius with 5%carbon dioxide. Within 24 hours post-transfection, transfer 50, 000 to 100, 000 cells per staining reaction into a suitable polystyrene round bottom tube. Wash the cells by adding one milliliter of staining buffer consisting of phosphate buffer saline, or PBS, with 0.5%fetal bovine serum.

Centrifuge at 300g at four degrees Celsius for five minutes. From this point onward, keep the cells on ice and work with cooled reagents. Prepare a master mix containing the required fluorophore conjugated antibodies required for staining.

Dilute the antibodies as required in a final staining volume of 50 microliters. After centrifugation of the cells, discard the supernatant thoroughly by pipetting off residual liquid without disturbing the cell pellet. Resuspend each pellet in 50 microliters of the prepared antibody mix.

Incubate the tubes at four degrees Celsius in the dark for at least 30 minutes. Wash the cells by adding one milliliter of staining buffer directly to each tube. Centrifuge at 300g at four degrees Celsius for five minutes and discard the supernatant thoroughly by pipetting off residual liquid.

Resuspend the cell pellets in 100 microliters of staining buffer per tube. Finally, before starting the measurement, add two microliters of 7-Aminoactinomycin D solution to each tube and mix well. Incubate for 10 minutes at room temperature in the dark and proceed immediately to fluorescence measurement.

The viability of transfected chimeric antigen receptor, or CAR, T-cells ranged from 51 to 85%and did not differ significantly between CD3/28 bead and TransAct-activated T-cells. The highest frequency of CAR-positive cells was detectable within 24 hours after electroporation. The signal intensity of CAR expression was stable within the first six hours after electroporation and gradually decreased thereafter.

CAR expression was almost absent after 48 hours, demonstrating the transient nature of cell manipulation and the importance of choosing an early time window for downstream assays. The CD4 positive to CD8 positive ratio did not differ between CAR and MOCK transfected T-cells for both activation reagents. CAR expression did not alter the T-cell phenotype compared to MOCK transfected cells.

Electroporation width as little as 0.1 micrograms of mRNA led to a detectable mutant green fluorescent protein signal after 24 hours. Signal intensity increased, with increasing mRNA amounts up to one microgram, while three in six micrograms did not further increase the signal. A key challenge is donor-dependent variability in primary T-cells.

Therefore, sufficient biological replicates are essential to ensure reproducibility and reliable interpretation of experimental results. This protocol can be combined with CAR T-cell efficacy analysis, including cytotoxicity, or repeated stimulation assays, or the measurement of circulated cytokines for broader functional evaluation.