Two-stage microchamber array chip for single-cell clonal culture and drug-resistance assays

Owing to significant interpatient differences in anticancer agent sensitivity and intratumoral heterogeneity at the single-cell level, rapid evaluation and selection of anticancer agents tailored to individual patients are desirable. In this study, we propose a two-stage microchamber array chip (TMC) for high-density single-cell trapping, clonal culture, and functional drug-response observation. The proposed geometry was designed to balance efficient single-cell occupancy, clonal expansion, and suppression of overproliferation within densely arranged microchambers. Comparative evaluation of multiple microchamber geometries demonstrated that the two-stage structure improved the generation of independent single-cell-derived colonies while maintaining practical single-cell occupancy. As a proof-of-concept application, human lung cancer PC9 cells were cultured in media containing different concentrations of gefitinib. The proportion of proliferating monoclonal colonies decreased with increasing anticancer agent concentration, demonstrating the feasibility of observing drug-response-associated clonal behavior within the proposed platform. These results establish the utility of the TMC as a single-cell culture platform and suggest its potential applicability to future single-cell drug-response studies.