Advancements in 3-D in vitro models of the gastrointestinal system are leading to their adoption as reliable tools for toxicology, drug validation, and disease modeling. 3-D in vitro models, such as organoids and spheroids, provide a more accurate representation of the actual tissue microenvironment when compared to 2-D cell culture models. Some of the major issues encountered with current 3-D gastrointestinal models include variability, tissue viability, and experimental accessibility. To overcome these obstacles, we introduce MimEX™ GI, an innovative human “open book” gastrointestinal tissue model system. This system utilizes the unique characteristics of adult “ground-state” (GS) stem cells to generate 3-D gastrointestinal epithelial tissue on a 2-D surface. Using this technology, GS stem cells can be isolated from the epithelium of any region of the adult gastrointestinal tract and clonally expanded in 2-D culture.

Subsequently, using MimEX GI Differentiation Media and Reagents, a high-density monolayer of region-specific gastrointestinal stem cells differentiate into a 3-D tissue mimicking their respective tissue of origin ex vivo. The differentiated tissue obtained is uniform and oriented such that the apical surface of the mucosa is accessible to experimental manipulation. Here we characterize GS stem cells and their differentiated derivatives using a variety of techniques including immunostaining, Simple Western™, Single-Cell Western (Milo™), and RNAscope® in situ hybridization to analyze the protein and RNA expression of the GS stem cells post-isolation and differentiation. Additionally, MimEX GI tissue is used in a high-throughput permeability assay. The results obtained demonstrate the barrier function ability of ex vivo-generated tissue and its response to different molecules, demonstrating the flexibility of this tissue in high-throughput drug validation.