Enrichment of T-lymphocytes from leukemic blood using inertial microfluidics toward improved chimeric antigen receptor-T cell manufacturing

Chimeric antigen receptor (CAR-T) cell therapy is a successful immunotherapy for treatment of blood cancers. However, hurdles in their manufacturing remain including efficient isolation and purification of the T-cell starting material. Herein, we describe a one-step separation based on inertial spiral microfluidics for efficient enrichment of T-cells in B-cell acute lymphoblastic leukemia (ALL) and B-cell chronic lymphocytic leukemia (CLL) patient's samples. In healthy donors used to optimise the process, the lymphocyte purity was enriched from 65% (SD±0.2) to 91% (SD±0.06) and T-cell purity was enriched from 45% (SD±0.1) to 73% (SD±0.02). Leukemic samples had higher starting B-cell compared to the healthy donor samples. Efficient enrichment and recovery of lymphocytes and T-cells was achieved in ALL samples with B-cells, monocytes and leukemic blasts depleted by 80% (SD±0.09), 89% (SD±0.1) and 74% (SD±0.09) respectively, and a 70% (SD±0.1) T-cell recovery. CLL samples had lower T-cell numbers, and the separation process was less efficient compared to the ALL. This study demonstrates the use of inertial microfluidics for T-cell enrichment and depletion of B-cell blasts in ALL, suggesting its potential to address a key bottleneck of the CAR-T manufacturing workflow.