Animal-Free Intestinal Epithelial 2D "Organoid-Sheet" Cultures Using Invasin

The intestinal epithelium consists of a single layer of polarized cells that serves as a crucial barrier against pathogens while mediating food digestion and nutrient absorption. The differentiated villus cells, important for the intestine's function, originate from rapidly dividing Lgr5⁺ stem cells located in the intestinal crypts. These stem cells can be expanded in vitro and form three-dimensional (3D) "mini-organs" known as intestinal organoids, which closely phenocopy the intestinal epithelium. The development and maintenance of these 3D organoids depend on a defined growth factor cocktail and on extracellular matrix (ECM) support to preserve apical-basal polarity - an essential feature for nutrient uptake, growth, and the barrier function. Matrigel or BME has been used as the standard ECM hydrogel scaffold for organoid culture. Recently, we identified the bacterial protein Invasin as a defined ECM substitute that can fully recapitulate Matrigel/BME function. Utilizing recombinant Invasin coated on transwells, we developed a two-dimensional (2D) intestinal monolayer culture system. The intestinal stem cells form a polarized, confluent epithelial sheet while maintaining stemness and differentiation capacity into functional epithelial cell types. Compared to traditional 3D cultures, this 2D system offers advantages such as improved imaging, easier access to apical and basal compartments, and compatibility with automation for high-throughput applications. Here, we present a detailed protocol for generating 2D intestinal monolayers using Invasin, starting from either fresh human intestinal tissue or from pre-established 3D organoids. These 2D Invasin-cultures recapitulate key aspects of intestinal epithelial biology and are amenable to a broad range of experimental manipulations, including studies on gene function, disease modeling, pathogen interaction, co-cultures, and cell fate determination. These defined 2D-Invasin "organoid-sheets" cultures support the realization of clinical applications using healthy and patient-derived intestinal epithelial cells.