Intestinal organoid coculture systems: current approaches, challenges, and future directions

The intestinal microenvironment represents a complex and dynamic ecosystem, comprising a diverse range of epithelial and nonepithelial cells, a protective mucus layer, and a diverse community of gut microbiota. Understanding the intricate interplay between these components is essential for uncovering the mechanisms underlying intestinal health and disease. The development of intestinal organoids, three-dimensional (3-D) mini-intestines that closely mimic the architecture, cellular diversity, and functionality of the intestine, offers a powerful platform for investigating different aspects of intestinal physiology and pathology. However, current intestinal organoid models, mainly adult stem cell-derived organoids, lack the nonepithelial and microbial components of the intestinal microenvironment. As such, several coculture systems have been developed to coculture intestinal organoids with other intestinal elements including microbes (bacteria and viruses) and immune, stromal, and neural cells. These coculture models allow researchers to recreate the complex intestinal environment and study the intricate cross talk between different components of the intestinal ecosystem under healthy and pathological conditions. Currently, there are several approaches and methodologies to establish intestinal organoid cocultures, and each approach has its own strengths and limitations. This review discusses the existing methods for coculturing intestinal organoids with different intestinal elements, focusing on the methodological approaches, strengths and limitations, and future directions.