Microfluidic single-cell culture represents a versatile approach for tumor stem cell expansion and tumor organoid...

Tumor stem cells (TSCs) play a pivotal role in the development of tumor organoids. Consequently, the development of effective methods for the isolation and differential induction of TSCs is essential for the establishment of tumor organoids. In this study, we demonstrate a microfluidic single-cell culture technique that facilitates the selective expansion of TSCs and the subsequent generation of tumor organoids. Our findings demonstrate that microfluidic single-cell culture enables the generation of single-cell-derived tumorspheres (SDTs) across a variety of tumor cell lines of various tissue origins. The SDT cells exhibited definitive stem cell characteristics, as confirmed by the expression of stemness markers and functional cellular assays. Furthermore, the differential induction of individual TSCs resulted in the formation of single-cell-derived tumor organoids (STOs). The suitability of a microfluidic single-cell culture approach for patient-derived tumor specimens was also evaluated. Specifically, TSCs were successfully expanded from 16/26 primary colorectal cancer specimens, with SDT formation rates ranging from 0.02% to 17.77%. Differential induction culture of individual TSCs yielded enhanced STO formation efficiencies (25.02% to 65.30%). Collectively, these results establish microfluidic single-cell culture as a robust and adaptable methodology for TSC expansion and tumor organoid generation, offering a valuable platform to advance the field of tumor organoid engineering.