An Ex Vivo Technique to Generate Tumor-Specific Chimeric Antigen Receptor T Cells
Transkript
Take retroviral vectors mixed with primary spleen cells.
The vectors contain transgenic RNA encoding a chimeric antigen receptor or CAR, which targets a tumor-specific antigen.
The cytokine interleukin-2 in the media selectively promotes T cell proliferation while the other cells eventually die.
Transfer the mixture to a microplate coated with recombinant fibronectin fragments.
Centrifuge to facilitate the fibronectin fragments binding the vectors and T cells, mediating virus-host cell interaction, and incubate.
Upon fusion of the vectors' envelope with the cell membrane, the viral enzymes reverse-transcribe the released RNA into DNA and incorporate it into the host genome.
The transduced T cells express surface-bound CARs, consisting of an extracellular antibody variable fragment targeting the tumor-specific antigen and intracellular signaling regions to respond to the antigen.
Resuspend the cells before transferring them to a tube.
Centrifuge to discard the supernatant containing non-internalized viruses, and resuspend the cells in a buffer for downstream assays.
