An Ex Vivo Technique to Manufacture Chimeric Antigen Receptor T Cells
Transcription
Take primary T cells, mixed with antibody-conjugated magnetic beads in an interleukin-2-supplemented medium, and incubate.
The antibodies bind to CD3 in the T cell receptor complex and the co-stimulatory receptor CD28, while interleukin-2 binds to its cognate receptor, causing T cell activation and proliferation.
Introduce lentiviral vectors with a transgenic RNA encoding a chimeric antigen receptor or CAR, and incubate.
Upon vector-cell membrane fusion, the viral RNA undergoes reverse transcription into DNA, integrating into the host genome.
The transduced T cells express surface-bound CARs.
Harvest the cells at defined intervals.
For cryopreservation, take an aliquot of cells, detach the bound beads by pipetting, and magnetically separate them.
Spin down the cells and resuspend them in a cryopreservation medium.
Freeze the cells and store them at a cryogenic temperature.
To assess CAR expression, take another aliquot of cells, spin down the cells, and resuspend them in a buffer.
Introduce a CAR-specific fluorescently-tagged antibody and detect CAR expression using flow cytometry.
