A simple yet effective microfluidic device for thein-situformation of uniform-sized cell-laden microgels

Alginate microgels are attractive platforms for cell encapsulation, yet conventional gelation strategies often lead to heterogeneous crosslinking, unstable droplets, and reduced cell viability. Here, we present a paraffin oil-based flow-focusing microfluidic system that integratesin situandex situgelation to generate structurally homogeneous and monodisperse Ca-ALG microgels. Unlike conventional approaches that often suffer from unstable droplet formation or incomplete gelation, our method reliably produced uniform microgels with coefficients of variation consistently below 5% and maintained spherical morphology across a wide range of flow conditions. Scanning electron microscopy revealed a hierarchical porous architecture that supported nutrient and metabolite transport while providing structural stability. Encapsulated HEK-293 cells remained highly viable for more than two weeks, and spontaneous spheroid formation occurred within 24 h-an outcome rarely achieved in comparable systems and underscoring the functional relevance of this platform. Compared with existing microfluidic methods, this paraffin oil-driven dual gelation strategy offered superior reproducibility, droplet stability, and encapsulation efficiency. This study integrates and optimizes previously reported dual gelation strategies by employing paraffin oil in a flow-focusing device, establishing a simple, practical, and scalable solution to long-standing challenges in microgel-based encapsulation with strong potential to advance 3D culture, tissue engineering, and regenerative medicine.