A pump-free microfluidic co-culture system for investigating NK cell-tumor spheroid interactions in flow conditions

Natural killer (NK) cells are pivotal in immunotherapy due to their potent tumor-targeting capabilities. However, accessible in vitro 3D dynamic models for evaluating Tumor Infiltrating Natural Killer Cells (TINKs) remain scarce. This study addresses this gap by developing a novel pump-free microfluidic chip to investigate the interactions between NK-92 cells and prostate DU 145 tumor spheroids. The platform facilitates the separation of free NKs and TINKs for subtype characterization. The design integrates multiple planes with a multi-layer paper scaffold to accommodate tumor spheroids, allowing NK-92 cells to traverse Matrigel-coated barriers that mimic the extracellular matrix. The dual-channel pump-free device enables unidirectional circulation of NK-92 cells, allowing analysis of tumor spheroid movement and NK-92 cell interactions under flow conditions. Results demonstrate continuous fluid circulation in the dual-channel device by rocking the platform at tilt angles of 21° and 15°. Tumor spheroids show- enhanced migration under flow conditions compared to static culture. Although spheroids recruit slightly more NK-92 cells under flow conditions, CD56 and CD16 receptor expression on IL-2-activated free NK-92 cells and tumor-infiltrating NK-92 cells matches in vivo patterns in dynamic cultures. These findings suggest that tumor cells and fluid dynamics significantly influence NK cell subtypes. This pump-free microfluidic platform is a functional tool for simulating and studying immune cell-tumor interactions, providing valuable insights into NK cell dynamics with tumor spheroids in physiologically relevant environments.