A Microwell Array Embedded Microfluidic Gradient Platform for Drug Screening on Tumor Spheroids

ABSTRACT Microfluidic gradient platforms, especially flow‐based types, have emerged as promising tools in 3D tumor spheroid research, as they enable precise control of concentration gradients with rapid establishment and sustained long‐term stability. However, current flow‐based gradient chips are often constrained by limited spheroid culture capacity, restricting their utility for large‐scale drug evaluation. Here, we developed a microfluidic gradient platform embedded with high‐density microwell arrays (MEG platform), which enables high‐throughput and physiologically relevant spheroid culture across multiple chambers on a single chip. The total number of spheroids is flexibly tuned by adjusting the microwell array sheet size, and both monoculture and co‐culture spheroids maintain continuous and stable growth with well‐defined morphology. The platform demonstrates stable gradient delivery under dynamic perfusion. It is further applied to assess drug responses in monoculture and co‐culture spheroids, with co‐cultures exhibiting enhanced resistance compared with monocultures. The high‐throughput MEG platform should facilitate the use of tumor spheroid models in large‐scale drug testing and tumor‐stromal interaction studies.