Gut-on-chip methodology based on 3D-printed molds: a cost-effective and accessible approach

Gut-on-chips (GoC) represent a disruptive and promising technology to unravel the underlying mechanisms of gut health and pathology in physiologically relevant contexts. Researchers aiming to adopt this approach typically face a choice between purchasing expensive commercial microfluidic chips or building custom devices from scratch in their laboratories. However, designing such microfluidic systems requires specialized technical skills. Moreover, fabricating the master molds used in chip production is both costly and time-consuming, often requiring access to cleanroom facilities and advanced microfabrication equipment. Consequently, widespread adoption of GoC technology in biology and health research laboratories remains limited due to these technological and economic barriers. To address these challenges, we present the 3DP-μGut, an open-access, low-cost gut-on-chip platform fabricated using a standard desktop stereolithography (SLA) 3D printer. The device design is simple and user-friendly, making it accessible to a broad range of laboratories. The method has been optimized to allow the batch production of multiple chips with reproducible quality, suitable for biological experimentation. Furthermore, the device was improved to support high-resolution confocal live imaging and is compatible with various microfluidic pump systems, from basic to fully integrated setups. As a proof of concept, Caco-2 cells were cultured in the 3DP-μGut, and after 7 days of maturation, the cells formed a self-organized 3D epithelium mimicking <i>in vivo</i> structures. Finally, to showcase the system's versatility, <i>Lactiplantibacillus plantarum</i> and <i>Shigella flexneri</i> were introduced to demonstrate coculture and infection assays, respectively. This work highlights 3D printing as a powerful enabler of affordable, customizable GoC platforms.