An Accessible Microfluidic Platform for the Generation of Polarized Brain Organoids

ABSTRACT The integration of microfluidic technologies in organoid generation has enhanced the development of precise and functional 3D cell culture models, overcoming the limitations of traditional cultures, especially for brain organoids. The application of controlled morphogen gradients in microfluidic platforms has enabled faithful tissue patterning, cellular differentiation, and spatial organization of brain organoids. Here, we report the design, fabrication, and validation of a custom‐made double‐gradient microfluidic device for organoid culture. The device consists of a central channel for organoid growth, flanked by two parallel lateral channels that serve as reservoirs. These components were assembled using a porous membrane as an interface, bonded using APTES surface treatment and corona discharge activation, followed by thermal curing to ensure stability. Human embryonic stem cells were seeded and labeled with fluorescent dyes, where asymmetric fluorescence intensity confirmed efficient double‐gradient formation. Subsequently, midbrain organoids were loaded into the device under the same conditions, demonstrating gradient penetration into tissue‐level structures. A polarized differentiation protocol was successfully established within the platform, leading to the generation of differentiated dorsal and ventral neurons with asymmetric spatial distribution. These results establish a robust and reproducible pump‐free protocol for a double‐gradient microfluidic device, providing a versatile tool to establish polarized organoids for studies in patterning or the incorporation of more functional organoids in damaged tissues.