ORGANOTYPIC HUMAN SMALL INTESTINAL TISSUE TO ASSESS EPITHELIAL RESTITUTION.

Purpose:  The intestinal epithelium (SMI) forms an important physiological barrier in the gastrointestinal tract but this barrier can be compromised by a wide range of substances including drugs, microbes, and dietary substances traveling through the lumen.  Since repeated epithelial damage or injury are implicated in intestinal disorders including inflammatory bowel diseases, rapid closure or resealing of wounds has key physiological importance.  In this study, we describe an in vitro SMI model cultured using normal human intestinal cells which closely recapitulates the physiology and function of the small intestine to study epithelial restitution. Methods:  Normal human primary SMI epithelial cells, fibroblasts, and endothelial cells were expanded in monolayer culture and seeded onto transparent microporous membrane inserts to reconstruct the 3D SMI tissues. Injury was induced on the 3D tissues using 1 or 2 mm biopsy punches.  The injured tissues were analyzed daily for epithelial restitution using phase contrast microscopy, confocal imaging of migrating epithelial cells (immuno-stained for cytokeratin 19) and fibroblasts (immuno-stained for vimentin), transepithelial electrical resistance (TEER) measurements to monitor recovery of epithelial barrier integrity, and H&E staining to examine the level of wound closure and re-epithelialization. Results:  Following injury, TEER of the SMI tissues dropped from 160 Ω*cm2 (baseline) to 55 Ω*cm2.  On day 1 after the injury, fibroblasts became more visible in the wound area and epithelial cells shouldering the wound began to migrate into the wounded area. Confocal and H&E imaging of injured tissues showed cooperation of fibroblasts and epithelial cells in the wound healing process.  Wounded areas not resealed by epithelial cells were initially covered by fibroblasts.  Overall, completion of wound healing was achieved in 4-6 days post-injury. On days 4-6: 1) the migrating epithelial cells resealed the wound and migrating epithelial cells re-polarized (confirmed by confocal imaging and H&E staining) and 2) tissue barrier returned to pre-injury, baseline levels (TEER). Conclusions:  The newly developed SMI tissue model will likely be useful for testing candidate drugs or biologics to treat diseases that are characterized by injuries of small intestine epithelial barrier.

Brush borders, Cell migration, Claudin-1, Cytokeratin 19, DAPI, EpiIntestinal (SMI-100-FT, SMI-100), Epithelial restitution, Inflamed bowel disease, TEER, Tight junctions, Vimentin, Wound healing, ZO-1

Punch biopsy