Efficient and Rapid Generation of CAR-T and Cytokine-Induced Killer Cells in GMP-scalable Devices

Adoptive cell therapies (ACT) have progressed rapidly in recent years, playing a pivotal role in cancer immunotherapy and significantly influencing treatment outcomes. A critical step in cell therapy development, at both preclinical and clinical stages, is the manufacturing process. Conventional static culture methods require frequent cell manipulation and are often limited by reduced gas exchange and nutrient supply, which can compromise cell yield and phenotype. Here, we present a step-by-step, reproducible, and scalable protocol for the ex vivo expansion of two ACT products, namely Chimeric Antigen Receptor (CAR)-T and Cytokine-Induced Killer (CIK) cells, using Gas-permeable Rapid expansion (G-Rex) devices. G-Rex devices are specifically designed to enhance nutrient exchange and support high-density cultures with minimal user intervention. Among various ACT approaches, CAR-T cells have demonstrated remarkable success in treating hematological malignancies, facilitating a rapid advance from experimental research into clinical applications. In addition to CAR-T cells, CIK cells have also been widely applied in clinical trials due to their characteristic phenotype and the absence of treatment-related adverse events and Graft-versus-Host Disease (GvHD). This protocol outlines the key technical steps for both cell types, starting with the isolation and seeding of peripheral blood mononuclear cells (PBMCs). Briefly, PBMCs are stimulated with anti-CD3/CD28 antibodies, followed by lentiviral transduction to expand CAR-T cells, or with Interferon-γ (IFN-γ), anti-CD3 antibody, and Interleukin 2 (IL-2) to obtain CIK cells. Flow cytometry is employed to monitor the viability and phenotype of the cells, and functional assays are performed to confirm the therapeutic potential of the cell product and to validate the scalability of the approach. Overall, this protocol provides a practical solution for producing large numbers of effector cells in a preclinical setting, facilitating clinical application with several immune cell populations. Finally, it offers a scalable solution for high-yield effector cell manufacturing.