Robust bioengineered 3D functional human intestinal epithelium

Abstract

Intestinal functions are central to human physiology, health and disease. Options to study these functions with direct relevance to the human condition remain severely limited when using conventional cell cultures, microfluidic systems, organoids, animal surrogates or human studies. To replicate in vitro the tissue architecture and microenvironments of native intestine, we developed a 3D porous protein scaffolding system, containing a geometrically-engineered hollow lumen, with adaptability to both large and small intestines. These intestinal tissues demonstrated representative human responses by permitting continuous accumulation of mucous secretions on the epithelial surface, establishing low oxygen tension in the lumen, and interacting with gut-colonizing bacteria. The newly developed 3D intestine model enabled months-long sustained access to these intestinal functions in vitro, readily integrable with a multitude of different organ mimics and will therefore ensure a reliable ex vivo tissue system for studies in a broad context of human intestinal diseases and treatments.

Novel Bioengineered Three-Dimensional Human Intestinal Model for Long-Term Infection of Cryptosporidium parvum

“…To our knowledge, this study demonstrates the longest continuous C. parvum infection in a static in vitro culture model. In addition, although this model has been shown to support infection by the bacterial pathogen Yersinia pseudotuberculosis and coculture with the commensal Lactobacillus rhamnosus (26), this is the first report of infection by a protozoan pathogen in this bioengineered 3D model. This 3D model has specific features not present in previously reported 2D models which may contribute to the longer duration of infection.…”

“…A major advantage of the 3D system over 2D models is the ability to generate depth-graded oxygen profiles in the luminal direction, with the lowest oxygen tensions being identified in the most distal regions, which recapitulates the proximal regionto-distal region decrease in luminal oxygen tension along the gastrointestinal tract (26). This is important, since Cryptosporidium is a microaerophilic/anaerobic organism (32) which preferentially colonizes the terminal ileum, which has a low-oxygen environment.…”

“…Two types of intestinal epithelial cells which are routinely used in coculture for intestinal model systems (30), cells of the Caco-2 and the HT29-MTX cell lines, which are differentiated for 2 weeks in situ, are included, resulting in enterocyte-like and goblet cell-like cells, respectively. These cells grow as highly polarized monolayers characterized by microvilli with continuous brush borders and the formation of tight junctions, identified by zonula occludens protein 1 (ZO-1) immunostaining (26), in the scaffold lumen. Biomarkers of the functional activity of enterocytes, such as alkaline phosphatase and sucrose isomaltase, as well as robust mucus secretion, identified by MUC-2 immunostaining and alcian blue staining, confirm that this model recapitulates human intestinal epithelial cell function.…”

“…3D silk scaffolds were prepared as described previously (26). Briefly, silk fibroin was extracted from Bombyx mori silkworm cocoons.…”

“…Recently, we developed a novel bioengineered three-dimensional (3D) human intestinal tissue model using silk protein as the scaffolding biomaterial (26). This 3D porous protein scaffolding system contains a geometrically engineered hollow lumen with adaptability to both the large and small intestines.…”