A macrophage-intestinal organoid co-culture model captures balanced epithelial homeostasis and inflammation.

Immune-epithelial interactions are essential for intestinal homeostasis, yet organoid monocultures lack immune components and fail to model how immune cells shape epithelial identity. Here, we establish a macrophage-organoid co-culture integrating bone marrow-derived macrophages (BMDMs) into the intestinal niche to create a controlled, immune-competent system. Under homeostatic conditions, the optimized 5k configuration of BMDMs preserves crypt-like architecture and maintains stem cell localization and secretory lineage organization. TNFα stimulation induces coordinated epithelial remodeling characterized by reduced canonical stem cell marker expression and density-dependent shifts in macrophage phenotype. Transcriptomic profiling confirms enrichment of immune-associated and epithelial stress pathways. Comparative analysis with a DSS-induced murine inflammation model reveals overlapping inflammatory and epithelial remodeling features, supporting physiological relevance. This platform provides a reproducible framework for studying epithelial-immune interactions and inflammatory remodeling under defined conditions, offering a controlled alternative to animal models.