Studies on cellular heterogeneity have emerged as a powerful approach for developing new strategies to treat diseases including cancer. However, it is difficult to set up an in vitro co-culture experiment to study the interaction of individual live cells. In this paper, we report a hydrodynamic shuttling chip (HSC) which can deterministically capture single cells into microfluidic chambers to set up multiple single-cell co-culture experiments in which individual live cells can be microscopically observed. Using this chip device, we demonstrated a triple single-cell culture of oral squamous cell carcinoma and lymphatic endothelial cells to observe the effect of cell-cell interaction on the cell motility. Triple, single-cell pairing efficiency by our HSC device was eightfold higher than that of the probabilistic method. Using this HSC device, we were able to perform triple-culture experiments to show the cell type-dependent motility of oral squamous cell carcinoma and lymphatic endothelial cells, which was not observed in co-culture experiments.