Dielectrophoretic high-purity isolation of primary T-cells in samples contaminated with leukemia cells, for biomanufacturing of therapeutic CAR T-cells

Abstract Chimeric antigen receptor (CAR) T-cell therapy is a Food and Drug Administration (FDA)-approved treatment for specific cancers. CAR T-cells are manufactured ex vivo using T-cells derived from patients. Recent clinical trials reported a very high level of efficacy as well as complete remission rates (70%–93%) for acute lymphoblastic leukemia (ALL) patients. However, there have also been reports of significant adverse effects resulting from the contamination of T-cells derived from patients with existing cancer cells during manufacturing. The accidental transfection of cancer cells with CAR genes provides the cancer cells with immunity from CAR T-cells. As a result, genetically modified cancer cells that express the CAR molecules grow into secondary tumors. This significantly undermines the great benefits offered by CAR T-cell therapy. The removal of cancer cells before gene transfer using cell purification methods can address this issue. However, traditional cell purification methods such as fluorescence-activated cell separation and magnetic-activated cell separation have proven to be unsuitable for high-purity T-cell purification. To address this issue, we investigated the utility, in microfluidic channels, of dielectrophoretic cell purification, which is a label-free high-throughput cell purification method. When we conducted the experiments with primary T-cell (CD8 + ) samples contaminated with cultured acute lymphoblastic leukemia and chronic myelogenous leukemia cells, we found that dielectrophoresis could be used to purify cell samples and achieve 100% purity with high cell viability (greater than 90%).