Rebuilding the gut: Decoding enteric pathogenesis using next-generation human in vitro models

Traditional in vitro and in vivo models often fail to replicate the complex physiology of the human gastrointestinal (GI) tract, and consequently, do not fully capture how this environment influences the infective strategies employed by enteric pathogens. Here, we evaluate air-liquid interface (ALI) cultures and gut-on-a-chip (GoC) microfluidic systems as next-generation in vitro models (NGIMs) that recreate critical features of the human GI mucosal surface, including the mucus layer, crypt-villus architecture, and biomechanical forces that modulate enteric pathogenesis. We review their application towards bacterial, fungal, viral, and parasitic infections, highlighting novel mechanisms that were previously undetected using traditional models. Comparing the two approaches, we outline how ALI excels for studies involving mucus and for cost-effective implementation, whereas GoC systems uniquely capture peristalsis and luminal flow, revealing dynamic pathogen-host interactions. Notably, both platforms enable complex co-culturing, allowing pathogen-host-commensal dynamics to be studied in a reductionist manner. Lastly, we discuss how future efforts aimed at better mimicking the human GI tract could incorporate stromal, immune, and enteric nervous system components, while also improving accessibility and standardization. Taken together, both ALI and GoC systems offer significant opportunities to provide new insights into infection biology, while also serving as effective preclinical platforms for precision medicine and the evaluation of next-generation antimicrobial therapies.