RECONSTRUCTION OF NEW ORGANOTYPIC 3D HUMAN SMALL INTESTINE TISSUE MODEL FOR DRUG SAFETY AND TOXICITY.

The epithelial lining of the gastrointestinal (GI) tract is a gatekeeper for entry of orally ingested nutrients and xenobiotics including medicaments. The small intestine has a well organized structure containing proliferative cells which migrate along the crypt-villi axis and differentiate into functionally mature epithelial cells. Currently, the most common in vitro model utilized for study of drug absorption in the small intestine is a 2-dimensional cell culture model cultured using the colon carcinoma cell line, Caco-2. These cells differentiate into monolayers of polarized enterocytes that are connected by tight junctions, but lack mucus-secreting goblet cells and show inter-passage inconsistency in the expression of drug transporters. Others have developed small intestine organoids which are not suitable for topical application of test articles. Here, we report the reconstruction of a human organotypic small intestine (SI) tissue model generated from primary human small intestinal (SI) epithelial cells that grow in tissue culture inserts using serum free medium. Normal (non-cancerous), human SI epithelial cells and myofibroblasts were expanded in monolayer culture and seeded on a microporous membrane containing a fibroblast-collagen-gel substrate to reconstruct 3-dimensional organotypic SI tissues. Tissue morphology, surface biomarker expression, and ultrastructural features of the SI model were characterized by H&E staining, immunohistochemistry, and transmission electron microscopy, respectively. Basal cell proliferation markers, cytokeratins (CK), mucin, and brush border membrane formation were monitored. Drug transporter expression was checked by RT-PCR. Tissue responses to inflammatory stimuli were examined following TNF-α exposure. Analysis of the SI tissue revealed: 1) wall-to-wall growth of the epithelial layer, 2) columnar epithelial cell morphology similar to that of native SI tissue, 3) expression of Mucin-2 (MUC-2), CK19, and villin at the surface of the epithelium, and 4) formation of brush borders and tight junctions. RT-PCR results showed expression of efflux drug transporter genes, MDR-1, MRP-1, MRP-2, and BCRP, and the metabolic enzyme, CYP3A4. Treatment of the Si tissue model with TNF-α induces a proinflammatory response by inducing the C-X-C chemokines such as IL-8 and GRO-α. In conclusion, the new human cell based 3D, SI tissue model will likely serve as a valuable tool to evaluate safety and adsorption of pre-clinical therapeutic drug candidates intended for oral administration as well as preclinical study of intestinal mucosal inflammation, microbiomes, and microbial infection mechanisms in the gastrointestinal microenvironment.

BCRP, brush border membrane,  Caco-2,  CYP3A4,  Cytokeratin-19,  Cytokeratin-8,  efflux drug transporters,  Gro-α,  IL-8,  Kerckring folds,  Lucifer yellow leakage,  MDR-1,  MRP-1,  MRP-2,  Mucin-2,  Mucous secretory granules,  P-glycoprotein,  Small intestine,  TEER, Villin

Lucifer yellow,  poly I:C, TNF-α