An Ex Vivo Technique to Generate Tumor-Specific Chimeric Antigen Receptor T Cells

Published: May 31, 2024

Abstract

Source: Riccione, K., et al. Generation of CAR T Cells for Adoptive Therapy in the Context of Glioblastoma Standard of Care. J. Vis. Exp. (2015).

This video demonstrates a technique for the ex vivo generation of tumor antigen-specific chimeric antigen receptor (CAR) T cells. Retroviral vectors carrying transgenic RNA are mixed with spleen cells in a medium supplemented with interleukin-2 to promote T-cell survival and proliferation. The mixture is then transferred to a plate coated with recombinant human fibronectin fragments and centrifuged to facilitate the fusion of the virus with the cell, allowing the RNA to integrate into the host genome and produce surface-bound CARs targeting the tumor antigen.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board. 1. Splenectomy and T Cell Preparation Day 0: Pour 10 ml of T cell media (TCM) into a 50 ml conical and place on ice for spleen collection. Sacrifice the appropriate number of animals by CO2 asphyxiation and secondary decapitation: Place animals in a cage receiving CO2</…

Açıklamalar

The authors have nothing to disclose.

Materials

Concanavalin A Sigma Aldrich C2010 Non-specific mitogen to induce T cell proliferation and viral transduction
RPMI 1640 Life Technologies 11875-093 T cell culture media
Bovine Serum Albumin (BSA), Fraction V—Standard Grade Gemini Bio Products 700-100P Blocks non-specific binding of retrovirus to retronectin-coated plates
Pharm Lyse (10X concentrate) BD Biosciences 555899 Lyses red blood cells during splenocyte processing
70 µm Sterile Cell Strainers Corning 352350 Filters away large tissue particles during splenocyte processing
100 mm BioCoat Culture Dishes with Poly-D-Lysine Corning 356469 Promotes HEK293 cell adhesion to maximize proliferation after transfection
Dimethyl Sulfoxide Sigma Life Sciences D2650 Necessary for complete dissolution of temozolomide
Saline Hospira IM 0132 (5/04) Solvent for temozolomide and ketamine/xylazine
Ketathesia HCl Henry Schein Animal Health 11695-0701-1 Ketamine solution
AnaSed Lloyd Inc N/A Xylazine sterile solution 100 mg/mL

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An Ex Vivo Technique to Generate Tumor-Specific Chimeric Antigen Receptor T Cells. J. Vis. Exp. (Pending Publication), e22238, doi: (2024).

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