摘要Multilayer paper-based cell culture,as an in vitro three-dimensional (3D) cell culture method,has been frequently used to research drug bioavailability,therapeutic efficacy,and dose-limiting toxicity in malignant tumors.This paper proposes a heterogenous multilayer paper stacking co-culture system to establish a model of natural killer (NK) cells moving through the endothelium layer and attacking tumor spheroids.This system consists of three layers:a bottom tumor-spheroid layer,a middle invasion layer,and a top endothelium layer.NK-92 cells were placed in the supernatant on top of the three layers.After two days of co-culture,the attack of tumor spheroids by NK cells was observed.We additionally examined the infiltration of NK-92 cells within the tumor spheroids at different Z-axis depths using a confocal microscope,and the results suggested that the system successfully realizes NK cells traveling cross the endothelium layer to form tumor-infiltrating NK cells (TINKs).The potential application of multilayer paper for co-culture models involving cancer cells and immune cells holds great promise for exploring the interaction dynamics of these two cell types.