Single-StepFabrication of V‑Shaped PolymericMicrowells to Enhance Cancer Spheroid Formation

Traditional cancer research has long relied on two-dimensional (2D) cell cultures, which inadequately mimic the complex three-dimensional (3D) microenvironments of <i>in vivo</i> tumors. Recent advancements in 3D cell cultures, particularly cancer spheroids, have highlighted their superior physiological relevance. However, existing methods for spheroid generation often require complex, multistep fabrication processes that limit scalability and reproducibility. In this study, we present a novel single-step photolithographic technique to fabricate high-aspect-ratio V-slanted hydrogel microwells. By employing polyethylene glycol (PEG)-based hydrogels, we create biocompatible, extracellular matrix (ECM)-like scaffolds that enhance gas and nutrient exchange while promoting uniform spheroid formation. The hydrogel microwells allow precise control of spheroid size, achieving a physiologically relevant diameter of 425 μm within 12–24 h, and the resulting spheroids exhibiting high viability over 3 weeks. Moreover, the method facilitates the creation of scalable multiwell arrays for high-throughput applications, making it suitable for both small-scale and large-scale experimental needs. This platform addresses the limitations of traditional microwell fabrication, offering a robust, efficient, and reproducible system for generating physiologically relevant 3D models with valuable applications in cancer research, drug testing, and tissue engineering.