A simple yet effective microfluidic device for the in-situ formation of uniform-sized cell-laden microgels

Abstract 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 integrates in situ and ex situ gelation 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.