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.

Free Epoxide Content Mediates Encapsulated Cell Viability and Activity through Protein Interactions in a Thermoresponsive, In Situ Forming Hydrogel.

The development of synthetic and well-defined extracellular matrices that are free of xenogeneic components and are capable of inducing desired cellular responses holds great potential for use in vitro as 3D cell culture environments and in vivo as scaffolds for tissue regeneration. In this study, the impact of free and partially occupied epoxide groups on the viability, activity, and behavior of rat fibroblasts encapsulated in thermoresponsive hydrogels based on poly(N-isopropylacrylamide) (pNiPAAm) was investigated. While fibroblasts encapsulated in neat pNiPAAm remained rounded and showed significant toxicity by 5 days, those encapsulated in the epoxide-modified thermogels demonstrated not only high viability but also an ability to proliferate, attach, produce extracellular matrix (ECM) components, and cluster. The results demonstrated that the presence of free epoxide groups led to the local conjugation of available proteins to produce a modified structure in situ, which supported cell viability, activity, and cluster formation within the hydrogel.

In situ spray deposition of cell-loaded, thermally and chemically gelling hydrogel coatings for tissue regeneration.

In this study, the efficacy of creating cellular hydrogel coatings on warm tissue surfaces through the minimally invasive, sprayable delivery of thermoresponsive liquid solutions was investigated. Poly(N-isopropylacrylamide)-based (pNiPAAm) thermogelling macromers with or without addition of crosslinking polyamidoamine (PAMAM) macromers were synthesized and used to produce in situ forming thermally and chemically gelling hydrogel systems. The effect of solution and process parameters on hydrogel physical properties and morphology was evaluated and compared to poly(ethylene glycol) and injection controls. Smooth, fast, and conformal hydrogel coatings were obtained when pNiPAAm thermogelling macromers were sprayed with high PAMAM concentration at low pressure. Cellular hydrogel coatings were further fabricated by different spraying techniques: single-stream, layer-by-layer, and dual stream methods. The impact of spray technique, solution formulation, pressure, and spray solution viscosity on the viability of fibroblast and osteoblast cells encapsulated in hydrogels was elucidated. In particular, the early formation of chemically crosslinked micronetworks during bulk liquid flow was shown to significantly affect cell viability under turbulent conditions compared to injectable controls. The results demonstrated that sprayable, in situ forming hydrogels capable of delivering cell populations in a homogeneous therapeutic coating on diseased tissue surfaces offer promise as novel therapies for applications in regenerative medicine. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2383-2393, 2016.