Fabrication of 3D engineered intestinal tissue producing abundant mucus by air-liquid interface culture using paper-based dual-layer scaffold.

Fabrication of engineered intestinal tissues with the structures and functions as humans is crucial and promising as the tools for developing drugs and functional foods. The aim of this study is to fabricate an engineered intestinal tissue from Caco-2 cells by air-liquid interface culture using a paper-based dual-layer scaffold and analyze its structure and functions. Just by simply placing on a folded paper soaked in the medium, the electrospun gelatin microfiber mesh as the upper cell adhesion layer of the dual-layer scaffold was exposed to the air, while the lower paper layer worked to preserve and supply the cell culture medium to achieve stable culture over several weeks. Unlike the flat tissue produced using the conventional commercial cultureware, Transwell, the engineered intestinal tissue fabricated in this study formed three-dimensional villous architectures. Microvilli and tight junction structures characteristic of epithelial tissue were also formed at the apical side. Furthermore, compared to the tissue prepared by Transwell, mucus production was significantly larger, and the enzymatic activities of drug metabolism and digestion were almost equivalent. In conclusion, the air-liquid interface culture using the paper-based dual-layer scaffold developed in this study was simple but effective in fabricating the engineered intestinal tissue with superior structures and functions.

Investigation on the Epoxidation of Piperitenone, and Structure-activity Relationships of Piperitenone Oxide for Differentiation-inducing Activity.

Piperitenone oxide, a major chemical constituent of the essential oil of spearmint, Mentha spicata, induces differentiation in human colon cancer RCM-1 cells. In this study, piperitenone oxide and trans-piperitenone dioxide were prepared as racemic forms by epoxidation of piperitenone. The relative configuration between two epoxides in piperitenone dioxide was determined to be trans by 1H NMR analysis and nuclear Overhauser effect spectroscopy (NOESY) in conjunction with density functional theory (DFT) calculations. Optical resolution of (±)-piperitenone oxide by high-performance liquid chromatography (HPLC) using a chiral stationary phase (CSP) afforded both enantiomers with over 98% enantiomeric excess (ee). Evaluation of the differentiation-inducing activity of the synthetic compounds revealed that the epoxide at C-1 and C-6 in piperitenone oxide is important for the activity, and (+)-piperitenone oxide has stronger activity than (-)-piperitenone oxide. The results obtained in this study provide new information on the application of piperitenone oxide and spearmint for differentiation-inducing therapy. Furthermore, natural piperitenone oxide was isolated from M. spicata. The enantiomeric excess of the isolated natural piperitenone oxide was 66% ee. Epoxidation of piperitenone with hydrogen peroxide proceeded in a phosphate buffer under weak basic conditions to give (±)-piperitenone oxide. These results suggest that the nonenzymatic epoxidation of piperitenone, which causes a decrease in the enantiomeric excess of natural piperitenone oxide, is accompanied by an enzymatic epoxidation in the biosynthesis of piperitenone oxide.