Efficient Capture and Traceless Release of Functional CD8⁺ T Cells with a Microfluidic Chip for Enhanced <i>In Vitro</i> and <i>In Viv</i><i>o</i> CD4-CAR Transduction

The chimeric antigen receptor (CAR) T cells targeting CD4 expressed cells in acute lymphoblastic leukemia (T-ALL) and acute myeloid leukemia (AML) could reduce the risk of off target effects in normal tissues. However, the efficacy of adoptive cell therapy is predominantly attributed to CD8⁺ T cells, necessitating their purification before lentivirus transfection to enhance the production of CD4-CAR-T cells. In this study, we developed a microfluidic chip functionalized with an optimized CD8 aptamer, A3t-MU, to facilitate the enrichment and purification of CD8⁺ T cells. The presented chip showed efficient capture and seamless release of CD8⁺ T cells from cultured T cells and peripheral blood mononuclear cells (PBMCs). The purity of the released CD8⁺ T cells reached 98.1%, representing a 13% improvement over the conventional magnetic bead separation method. CD4-CAR was efficiently transduced into the purified CD8⁺ T cells to construct CAR-T cells. We evaluated the antitumor capability of the CD4-CAR transduced CD8⁺ T cells (anti-CD4 CD8-CAR T cells) both <i>in vitro</i> and <i>in vivo</i>. The anti-CD4 CD8-CAR T cells exhibited significant cancer-cell-killing capacity across multiple tumor cell lines, including CEM, Jurkat, and MV4-11. Meanwhile, anti-CD4 CD8-CAR T cells significantly inhibited tumor growth <i>in vivo</i>. In conclusion, the presented microfluidic chip offers a cost-effective and high-purity approach for CD8⁺ T cell separation, enhancing CD4-CAR transduction and achieving efficient antitumor capability both <i>in vitro</i> and <i>in vivo</i>.