An Ex Vivo Technique to Manufacture Chimeric Antigen Receptor T Cells

Published: April 30, 2024

Abstract

Source: Ghassemi, S., et al. Manufacturing Chimeric Antigen Receptor (CAR) T Cells for Adoptive Immunotherapy. J. Vis. Exp. (2019)

This video demonstrates a technique to manufacture chimeric antigen receptor (CAR) T cells ex vivo. Human T cells are incubated with magnetic beads coated with anti-CD3 and anti-CD28 antibodies, along with a medium supplemented with interleukin-2 (IL-2), leading to T cell activation and proliferation. Transgenic lentiviral vectors are introduced, allowing the transgenic RNA to integrate into the host genome and express the transgene. This results in the transduced T cells expressing chimeric antigen receptors (CARs) on their surface. The T cells are harvested at defined time points to assess CAR expression and cryopreservation.

Protocol

All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines. 1. T Cell Activation, Transduction, and Expansion Activate fresh or cryopreserved primary human T cells by mixing with anti-CD3/CD28 magnetic beads (e.g., dynabeads) at a ratio of 3 beads per T cell in 6-well cell culture dishes. Culture T cells in X-VIVO 15 medium supplemented with 5% normal human AB serum, 2 mM L-glutamine, 20 mM 4-(2-hydroxyethyl…

開示

The authors have nothing to disclose.

Materials

Anti CD3/CD28 dynabeads Thermo Fisher 40203D
APC Mouse Anti-Human CD8 BD Biosciences 555369 RRID:AB_398595
APC-H7 Mouse anti-Human CD8 Antibody BD Biosciences 560179 RRID:AB_1645481
CountBright Absolute Counting Beads, Invitrogen C36950
HEPES Gibco 15630-080
Human AB serum Valley Biomedical HP1022
Human IL-2 IS, premium grade Miltenyi 130-097-744
L-glutamine Gibco 28030-081
Multisizer Coulter Counter Beckman Coulter
X-VIVO 15 Gibco 04-418Q

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記事を引用
An Ex Vivo Technique to Manufacture Chimeric Antigen Receptor T Cells. J. Vis. Exp. (Pending Publication), e22222, doi: (2024).

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