Dual Nozzle‐Assisted Deterministic Encapsulation of Triple Particles for Screening NK‐Cell Cytotoxicity Against Circulating Tumor Cell Clusters

Abstract Circulating tumor cell (CTC) clusters represent formidable precursors of cancer metastasis due to their heightened immune resistance against natural killer (NK) cells. Despite this, the cytotoxicity of NK cells against CTC clusters, particularly their interaction with other immune cells such as neutrophils, remains inadequately examined. This study introduces a dual‐nozzle integrated droplet microfluidic chip (dual‐nozzle chip) designed to facilitate the deterministic encapsulation of three distinct cell types—CTCs, NK cells, and neutrophils—to monitor the dynamic cytotoxicity between immune cells and target cells. The dual‐nozzle chip comprises double‐spiral channels and a serpentine channel for inertial cell focusing, alongside dual‐nozzle oil phases employed to generate monodisperse droplets at high flow rates. Utilizing Rayleigh–Plateau instability, the focused cell streams, characterized by high inertia, undergo pinching off into monodisperse droplets at the flow‐focusing junction, where dual‐nozzle oil phases are introduced. Consequently, triple cells are paired at the desired ratios, overcoming the intrinsic challenge posed by the Poisson distribution. A droplet‐based assay demonstrates that NK cell‐mediated cytotoxicity varies depending on the type of cancer cells and the presence of suppressor cells. The design strategy of the dual‐nozzle chip exhibits promises for broader applications, emphasizing its potential for analyzing diverse cell‐to‐cell interactions.