Hydrogel Confinement Strategies for 3D Cell Culture in Microfluidic Systems

ABSTRACT 3D hydrogel‐based cell culture systems are widely used to replicate native tissue microenvironments and study multicellular responses. In particular, microfluidic devices incorporating spatially confined hydrogel regions enable precise compartmentalization, dynamic perfusion, and multiplexed assay configurations. To achieve stable hydrogel localization within open channel networks, diverse confinement strategies have been developed, including micropillar arrays, capillary pinning via phaseguides, stepped‐height structures, porous membrane integration, and support‐free methods based on surface tension or sacrificial barriers. These designs allow open access to adjacent culture compartments, facilitating coculture and interface‐specific cellular interactions. This review categorizes hydrogel confinement strategies by structural principles and evaluates their advantages and limitations regarding geometric flexibility, ease of fabrication, and biological functionality. In addition, the fabrication methods used to implement these structures are discussed in detail, covering photolithography, 3D printing, micromilling for mold‐based soft lithography, and direct thermoforming of thermoplastic substrates. Representative commercial platforms are also introduced to demonstrate practical implementations. By integrating insights from structural design and manufacturing perspectives, this review provides a comprehensive guide for researchers to select or engineer 3D hydrogel culture chips tailored to diverse experimental demands in biomedical research and organ‐on‐a‐chip applications.