Spray Delivery of Intestinal Organoids to Reconstitute Epithelium on Decellularized Native Extracellular Matrix

The native extracellular matrix (ECM) serves as a unique platform for tissue engineering because it provides an organ-specific scaffold in terms of both matrix composition and tissue architecture. However, efficacious cell-seeding techniques for recellularizing the ECM constructs with appropriate cell types to restore biological function remain under development. In this study, the impact of spraying as a seeding technique for repopulation of decellularized small intestine was investigated. In a series of experiments, CaCo-2 cells were first used to investigate the effect of spray device type and pressure on cell viability and to optimize parameters for seeding intestinal epithelial cells. High cell viability and a homogeneous cell distribution were obtained when cell suspensions were sprayed through an airbrush at low pressure. Next, the effect of seeding method and spray pressure on the size and dispersal of intestinal organoids, a more complex and clinically relevant intestinal stem cell population, was evaluated. The feasibility of seeding intestinal epithelial cells onto decellularized scaffolds was next studied using sprayed CaCo-2 cells, which survived the spray-seeding process and formed a monolayer on the scaffold. Finally, airbrush seeding was used to spray intestinal organoids onto the scaffolds, with cell survival and tissue architecture evaluated after 1 week of culture. Organoids seeded through pipetting onto the decellularized scaffold survived, but demonstrated aggregation, with cells organized around multiple small lumens. In contrast, organoids airbrush spray seeded at 0.35 bar onto the decellularized scaffold not only engrafted but also demonstrated formation of an epithelial monolayer that resembled the absorptive surface found on intestinal villi. The results suggest that seeding cells through airbrush spraying holds great potential for use in tissue engineering, especially for large-scale tubular organ recellularization.