This article details a method for transducing murine CD8-positive T cells with a chimeric antigen receptor (CAR) using retroviral vectors. The process enhances gene transfer efficiency through the use of fibronectin-coated microwell plates.
Take a microwell plate coated with recombinant fibronectin fragments and add activated murine CD8-positive T cells.
Introduce retroviral vectors containing a transgenic RNA that encodes a chimeric antigen receptor or CAR.
Cover the plate and seal it in a bag to avoid contamination. Centrifuge the plate at a low speed to facilitate virus-cell interaction.
Fibronectin binds to the virus and the cell, colocalizing them to enhance virus-mediated gene transfer.
Post-centrifugation, remove the sealed bag and incubate the plate under physiological conditions.
The envelope of the viral vector fuses with the cell membrane, releasing the viral RNA and enzymes.
The viral enzymes reverse-transcribe the released RNA and integrate the resulting DNA into the host genome, enabling the cells to express surface-bound CARs.
The CAR comprises an antigen-binding domain specific to the target antigen and signaling domains for T cell activation.
The antigen-specific CAR T cells are ready for downstream assays.
On day 1, prepare human fibronectin fragment-coated plates by adding 0.5 milliliters of Fibronectin to the wells of a 24-well plate and incubating it overnight at 4 degrees Celsius.
On the next day, remove the fibronectin solution and add 1 milliliter of 2% BSA in PBS per well. Incubate the plate at room temperature for 30 minutes, and wash the treated wells with 1 milliliter of sterile PBS. The plate is ready to use after removing wash solution.
Count the activated CD8 T cells using trypan blue. Collect them by centrifugation, and resuspend them at 5 million viable cells per milliliter for transduction. Add 100 microliters of the activated CD8 cell suspension to each well of the fibronectin-coated plate. Then, add 1.5 to 2 milliliters of the previously prepared virus-containing medium, and mix using a swirling motion to evenly dispense the cells.
Seal the plate in a zip-lock bag and centrifuge it at 2000 times g for 90 minutes at 37 degrees Celsius. Remove the plate from the centrifuge and take it to a biological safety cabinet. Carefully remove the plastic bag and ensure that the outside of the plate is not contaminated with medium. Transfer the plate to a 37 degrees Celsius carbon dioxide incubator.
After 4 hours, remove 1 milliliter of medium from each well. Replace it with 1 milliliter of pre-warmed complete T cell medium and put the plate back in the incubator.
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