Efficient Generation of Murine Chimeric Antigen Receptor (CAR)-T Cells

Our research explores effective combinations of engineered bacteria and CAR-T cells for solid tumor treatment. It's important for us to study how these two cell therapies function within the context of a complete immune system using syngeneic models of murine cancer. Studying the function of CAR-T cells in syngeneic tumor models has been limited by the lack of standardized protocols for generating murine CAR-T cells.

Murine T-cells are particularly difficult to transduce and culture ex vivo using traditional methods. We developed this protocol to streamline the production of murine CAR-T cells to enable study in syngeneic models that provide important context to evaluate the activity of new CAR technologies. To begin, pipette 3.75 milliliters of reduced serum medium into a tube labeled A and dilute the media with 105 microliters of the transfection reagent.

Vortex the contents of the tube to mix well. Next, add 3.75 milliliters of the reduced serum medium into a tube labeled B.Dilute the expression plasmids with 90 microliters of enhancer reagent. Now add the contents of tube A to tube B and pipette the solution to mix well before incubation.

Carefully remove 10 milliliters of cell culture media from a prepared Phoenix-ECO plate. Then add the entire volume of the transfection mix to the plate dropwise. Incubate the cells at 37 degrees Celsius in an incubator.

Place MTCM viral harvest medium in a water bath at 37 degrees Celsius to preheat it. Now, tilt the Phoenix-ECO plate and pipette out the culture medium. Next, pipette 30 milliliters of the prewarmed viral harvest medium slowly into the tilted plate.

Place the cells back in the incubator. After 48 hours of transfection, harvest the viral supernatant. Filter it through 0.45 micrometer PVDF filters.

With the back of a sterile syringe, crush isolated murine spleens through a 70 to 100-micrometer cell strainer into a 50-milliliter conical tube. Then, wash the strainer with 5 milliliters of T-cell isolation buffer. After bringing the final volume of the cells to 50 milliliters, count the cells with the hemocytometer.

Pellet the cells by centrifugation for 10 minutes at 450 G at 4 degrees Celsius or at room temperature. Resuspend the cell pellet containing the splenocytes with the recommended T-cell isolation kit. Then, isolate the CD3-positive T-cells using negative selection.

Transfer the magnetically-separated isolate to a 15-milliliter conical tube. Check the cell count again. Centrifuge the isolated T-cells for 10 minutes at 450 G at 4 degrees Celsius.

Then, resuspend the cell palette in MTCM activation medium. Now add antibody-coated magnetic beads and interleukin-2 to the cell suspension to activate the T-cells. Incubate the cells at 37 degrees Celsius overnight.

On day zero, precoat non-treated, sterile, 24 well plates with 0.5 milliliters of human fibronectin transduction enhancer reagent. Store the plates at 4 degrees Celsius overnight. The next day, remove the transduction reagent from the wells.

Block the wells with an equal volume of sterile-filtered, BSA-supplemented PBS for 30 minutes at room temperature. After incubation, remove the BSA PBS and wash the wells with 0.5 to 1 milliliter PBS. Next, add 1 milliliter of the neat retrovirus to each pre-coded well.

Centrifuge the plate at 2000 G for 90 minutes at 32 degrees Celsius. Then, add 1 milliliter of the activated T-cells to each virally-loaded well. Centrifuge the plates again at 450 G for 10 minutes at 32 degrees Celsius.

Incubate the plate at 37 degrees Celsius overnight. On the fifth day, resuspend the cells to dissociate the activated T-cells from the antibody-coated beads. Place the cell suspension on a magnet for 30 seconds, then transfer the suspension into an ex vivo culture vessel.

Place the vessels in an incubator at 37 degrees Celsius before flow cytometric analysis. The use of T-cell isolation kit yielded T-cell purities of less than 98%before transduction. Reproducible CAR expression rates of 65 to 75%were achieved.

Ex vivo cultures with interleukins 7 and 15 led to a 15-fold by day 10 postactivation from a single spleen. Culture of the T-cells with the interleukins led to comparable CD8-positive and CD4-positive T-cell frequencies. After 10 days of ex vivo culture, the stem cell memory populations were observed to be preserved.