Chimeric antigen receptor (CAR) T-cell therapy is a promising and evolving immunotherapy approach for cancer treatment. In allogeneic CAR-T therapies, TCR + cells must be removed from the final cell product because of immunogenicity problems. It is accomplished through a negative affinity cell selection process where TCR + cells are affinity bound to a bead. The harvested TCR-cells are the product cells. A multidimensional acoustic standing wave field separates cell-bead complexes from free cells in an acoustic fluidized bed. The feed solution motion is normal to the primary acoustic field. Irrespective of the particle acoustic contrast, an interface between a dense suspension on the bottom and clear fluid on top develops in the field. We examine the physics behind the development of the interface and its subsequent motion. This motion influences the purity, scalability, and recovery of the TCR- cells. We present the effects of different acoustofluidic parameters, e.g., bead concentration, bead acoustic contrast factor, frequency, and flow rate on interface formation and its movement. Theoretical calculations and experimental results are discussed. The acoustic fluidized bed has been shown to give final purities of 99 + % of TCR- cells from a starting purity of 60%–70%, with 70 + % recoveries of TCR- cells.

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