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</…
Declarações
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).