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We aimed to produce an acellular human tissue scaffold with a view to test the possibility of recellularization with bone marrow stem cells to produce a tissue-engineered small intestine (TESI). Human small-bowel specimens (n ‫؍‬ 5) were obtained from cadaveric organ donors and treated sequentially with 6% dimethyl sulfoxide in hypotonic buffer, 1% Triton X-100, and DNase. Each small intestine (SI) piece (6 cm) was recellularized with EPCAM؉ and CD133؉ allogeneic bone marrow stem cells. Histological and molecular analysis demonstrated that after decellularization, all cellular components and nuclear material were removed. Our analysis also showed that the decellularized human SI tissue retained its histoarchitecture with intact villi and major structural proteins. Protein films of common extracellular matrix constituents (collagen I, laminin, and fibronectin) were found in abundance. Furthermore, several residual angiogenic factors were found in the decellularized SI. Following recellularization, we found viable mucin-positive goblet cells, CK18؉ epithelial cells in villi adjacent to a muscularis mucosa with ␣-actin؉ smooth muscle cells, and a high repopulation of blood vessels with CD31؉ endothelial cells. Our results show that in the future, such a TESI would be ideal for clinical purposes, because it can be derived from the recipient's own immunocompatible bone marrow cells, thus avoiding the use of immunosuppression. STEM CELLS TRANSLATIONAL MEDICINE 2013;2:307-315

Cell culture studies of enterocytes are important in many fields. However, there are difficulties in obtaining cell lines from adult human intestine, such as microbial contamination of cultures from the tissue samples, short life span of enterocytes, overgrowth of mesenchymal cells, etc. Various model used to obtain adult intestinal cell lines are very complex requiring use of feeder layer or gel matrices. The aim of this study was to establish a novel method for the simple and reproducible isolation of human enterocytes. Enterocytes were isolated from SI samples (n = 5) obtained from cadaveric donors using a mechanical procedure, and separation with immunomagnetic beads coated with anti-EpCAM antibodies. Light and electron microscopy, flow cytometry and immunocytochemistry techniques were used to characterize the isolated cells. Immunohistochemical staining of normal SB biopsies confirmed that the cell cultures maintained an in vivo phenotype as reflected in cytokeratin expression CK18, CK20 and expression of intestine-specific markers such as sucrase isomaltase and maltase glucoamylase. Furthermore, the cells strongly expressed TLR-5, 6, 7, 8 and 10 and several molecules such as CD40, CD86, CD44, ICAM-1 and HLA-DR which are important in triggering cell-mediated immune responses. This novel technique provides a unique in vitro system to study the biology of enterocytes in normal conditions as well as to study inflammatory processes in various small bowel disorders.

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