3D alginate hydrogel microspheres with uniform micro-structure for cell culture and CVB3 infection

Abstract Viral organisms characterized by elevated transmissibility and pathogenicity constitute a substantial public health risk. Three-dimensional (3D) cell culture systems better mimic the in vivo microenvironment than traditional two-dimensional (2D) models, offering significant potential for virological research. Therefore, a method for natural viral growth based on 3D cell culture need be developed. In this study, we developed an integrated microfluidic platform for the efficient generation of highly uniform alginate hydrogel microspheres (AHMs) encapsulating HeLa cells, enabling robust 3D cell culture and subsequent infection with Coxsackievirus B3 expressing enhanced green fluorescent protein (CVB3-eGFP). Our results demonstrate that AHMs support high cell viability and facilitated cell proliferation within a biomimetic 3D matrix. By systematically reducing the alginate concentration from 1.0% to 0.6%, we enhanced viral accessibility while maintaining microstructural integrity, thereby significantly improving CVB3-eGFP infection rates, as confirmed by fluorescence imaging and western blot analysis. This study establishes a tunable, reproducible, and physiologically relevant 3D model for studying virus–host interactions, with broad applications in antiviral drug screening and infectious disease modeling.