Optimization of Microfluidic Preparation for Calcium Alginate Microspheres Based on Response Surface Methodology

Although calcium alginate microspheres prepared by microfluidic methods have been widely investigated, the combined effects of dispersed-phase composition, continuous-phase properties, and surfactant combinations on microsphere formation remain insufficiently understood. In this study, a microfluidic system was established using sodium alginate as the aqueous phase, mineral oil as the continuous phase, and calcium chloride as the receiving phase. Response surface methodology was applied to model and optimize the key parameters involved in microsphere formation. The regression model showed good fit and reliable predictive ability. Under the optimized conditions, validation experiments yielded an average target-size fraction of 81.25%, which agreed well with the predicted value, demonstrating the feasibility and reproducibility of the optimized process. Biological evaluation showed that the optimized alginate microspheres exhibited no significant cytotoxicity toward BxPC-3 cells and maintained high cell viability, as confirmed by Live/Dead staining and CCK-8 assay. Confocal imaging further showed that the encapsulated BxPC-3 cells remained highly viable and formed three-dimensional clusters within the alginate matrix. These findings provide a reproducible microfluidic approach for generating calcium alginate microspheres with potential utility in cell encapsulation, three-dimensional culture, and related biomedical applications.