DEVELOPMENT OF DECELLULARISED HUMAN GUT AS A NATURAL 3D-PLATFORM FOR INTESTINAL BIOENGINEERING

BACKGROUND and AIMS: As one of the largest and most functionally complex organs of the human body, the intestine is vulnerable to many disorders, which are burdened by high social costs. Options to investigate these functions with direct relevance to the human condition remain severely limited when using conventional 2D cell cultures and animal models. The field of tissue engineering has been utilized extensively for developing biomaterial scaffolds for the repair of impaired biological tissue. However, a major opportunity exists to use them as in vitro 3D models for the study of gastrointestinal diseases. To do so, we here designed novel protocols to generate acellular 3D scaffolds by decellularising human intestine samples recapitulating the natural intestinal microenvironment. METHODS: Human intestinal tissue were obtained from healthy donors and dissected into either 10 x 5 mm cubes or 60 mm tubular segments. In order to identify the most optimal method, we performed and compared eight different decellularisation protocols involving three systems; high agitation, low agitation and mild rolling. The decellularisation efficiency was determined by histochemical analysis for the elimination of cellular and nuclear material, as well as preservation of extracellular matrix (ECM) components and microarchitecture. In addition, collagen and DNA quantification assays were performed to further investigate the effectiveness of the protocols. RESULTS: Initially, decellularisation protocols were stopped when the tissue became translucent with the dissolution of cells. All protocols were able to successfully decellularise the gut samples, as Haematoxylin and eosin (H&E), and Sirius Red (SR) staining revealed effective elimination of nuclear and cellular components, respectively. Furthermore, DNA quantification revealed a significant decrease (p<0.01) in DNA content (< 50 ng/mg) in 3 decellularisation protocols compared to native gut. ECM preservation within these 3 protocols was evaluated by immunohistochemistry for collagen I, III, IV, fibronectin and laminin. Two out of the three protocols revealed ECM microstructure preservation of all 4 layers of the gut wall. This was further confirmed with a collagen quantification assay. CONCLUSIONS: Our results present two innovative and effective protocols for the decellularisation of human gut. These human-derived scaffolds may represent an innovative platform for disease modelling, biomarker discovery and drug testing in gastrointestinal disorders. Further studies are underway to ascertain the interactions and biocompatibility of these scaffolds with cells derived from human gut.