Employing human intestinal organoids as advanced in vitro models for mechanistic studies on dietary ingredients.

Epidemiological studies show that dietary factors significantly impact human health, influencing both harmful and beneficial outcomes in the gastrointestinal tract. In vitro 2D models have been used to study these mechanisms but fail to replicate critical intestinal processes. Human intestinal organoids, which better mimic the cellular complexity of the human intestine, offer a promising alternative. However, challenges such as culture variability, passage-dependent heterogeneity, and lack of immune and vascular systems remain, requiring advances in organoid technology for dietary research. This review provides a framework for utilizing intestinal organoids to study dietary impacts on intestinal health markers. Key considerations are model selection, organoid construction, quality assessment, exposure regimens, and data analysis. In construction, stem cell origin, culture matrix, and medium composition are compared, with insights into how each affects organoid functionality. Quality assessment strategies, including morphology, passage-dependent changes, and cell-type marker expression, are discussed. Advancements in AI-based tools for imaging and data quantification are also highlighted. This review offers actionable insights and practical considerations to refine experimental approaches, improving the relevance and reproducibility of human intestinal organoid models in dietary research.