<strong>Trap-and-Drop Microwell Array for Single-Cell Culture and Deterministic Cell Pairing</strong>

To understand how individual cells interact with other cell types, cell-cell interactions must be investigated at the single-cell level. Here, we propose a novel microfluidic device, termed a Trap-and-Drop Microwell Array (TDMA), for single-cell culture and deterministic pairing of single cells. Deterministic single-cell pairing is achieved by sequentially trapping single cells in an electroactive microwell array using dielectrophoresis, fabricated at the ceiling of a microfluidic channel, and subsequently transferring the trapped cells into PDMS cell culture microwells located beneath each electroactive microwell. By separating the functions of microwells for cell trapping and cell culture, the system enables independent optimization of microwell dimensions for efficient trapping and stable cell culture. We successfully demonstrated long-term single-cell culture of U937 cells, and efficient pairing of single cells by sequentially trapping and dropping two different populations of U937 cells, achieving a pairing efficiency of 62%. This system offers promising applications for analyzing long-term cell-cell interactions.