A 3D hanging spheroid-filter plate for high-throughput drug testing and CAR T cell cytotoxicity assay

Tumour spheroids are widely used in immune cell cytotoxicity assays and anticancer drug testing, providing a physiologically relevant model replicating the tumour microenvironment. However, co-culture of immune and tumour cells complicates quantification of immune cell killing efficiency. We present a novel 3D hanging spheroid-filter plate that efficiently facilitates spheroid formation and separates unbound/dead cells during cytotoxicity assays. Optical imaging directly measures the cytotoxic effects of anti-cancer drugs on tumour spheroids, eliminating the need for live/dead fluorescent staining. This approach enables cost-effective evaluation of T-cell cytotoxicity with specific chimeric antigen receptors (CARs), enhancing immune cell-based assays and drug testing in three-dimensional tumour models. 3D Hanging spheroid-filter plate (3DHS-FP) comprises a 3D hanging dripper (top) and a spheroid filter plate (bottom). It is designed to streamline spheroid formation and the effortless separation of unbound and dead cells from tumor spheroids.