A Novel Bioreactor for Reconstitution of the Epithelium and Submucosal Glands in Decellularized Ferret Tracheas

Abstract

Tracheal grafts introduce the possibility to treat airway pathologies that require resection. While there has been success with engraftment of the surface airway epithelium (SAE) onto decellularized tracheas, there has been minimal advancement in regenerating the submucosal glands (SMGs). We designed a cost-effective open-system perfusion bioreactor to investigate the engraftment potential of ferret SAEs and murine myoepithelial cells (MECs) on a partly decellularized ferret trachea with the goal of creating a fully functional tracheal replacement. An air–liquid interface was also arranged by perfusing humidified air through the lumen of a recellularized conduit to induce differentiation. Our versatile bioreactor design was shown to support the successful partial decellularization and recellularization of ferret tracheas. The decellularized grafts maintained biomechanical integrity and chondrocyte viability, consistent with other publications. The scaffolds supported SAE basal cell engraftment, and early differentiation was observed once an air–liquid interface had been established. Lastly, MEC engraftment was sustained, with evidence of diffuse SMG reconstitution. This model will help shed light on SMG regeneration and basal cell differentiation in vitro for the development of fully functional tracheal grafts before transplantation.

Development and characterization of ferret ex vivo tracheal injury and cell engraftment model

“…To better understand the factors that influence the efficiency of cell engraftment, we assessed the differences in the transcription of cell adhesion related genes between Tomato+ cells seeded on a denuded tracheal graft and an ECM-coated cell culture dish. To this end, we removed the SAE from freshly excised tracheas using a series of SDS-containing detergent washes as described previously ( 24 ). Then, we engrafted 10 7 p6 Tomato+ primary surface airway basal cells into a 3-cm ferret tracheal segment using a rotating bioreactor method developed by our group previously for ferret tracheal explant cultures ( 24 ).…”

“…To this end, we removed the SAE from freshly excised tracheas using a series of SDS-containing detergent washes as described previously ( 24 ). Then, we engrafted 10 7 p6 Tomato+ primary surface airway basal cells into a 3-cm ferret tracheal segment using a rotating bioreactor method developed by our group previously for ferret tracheal explant cultures ( 24 ). After 18 h, the explants were rigorously washed with PBS to remove any cells that did not fully engraft, and then the luminal surface of the tracheas was brushed to collect Tomato+ cells.…”

“…(A) Experimental design: freshly harvested WT tracheas from adult ferrets were stripped of the native SAE by washing the lumen with 1% SDS. The grafts were then seeded with 10 7 p6 primary Tomato+ ferret surface airway basal cells using rotating bioreactor as described previously ( 24 ). After 18 h, the grafts were washed with PBS 3 times to remove non-adherent cells.…”

3D Bioprinting in Otolaryngology: A Review

“…Regarding respiratory epithelial cell growth, this is best achieved with an air-liquid-interface. [94,95] On the other hand, different culture conditions are required for neovascularization and promotion of cartilage maturation of the tracheal rings, pointing to the need to optimize unique bioreactor systems to promote multitissue development. Moreover, the need for neovascularization of in vitro cultured tissue limits the size and thickness of engineered constructs.…”

Orthotopic Ferret Tracheal Transplantation Using a Recellularized Bioengineered Graft Produces Functional Epithelia

“…In a recent publication, our group introduced a novel bioreactor capable of partially decellularizing the luminal compartment of a ferret trachea and of supporting implanted allogeneic surface airway epithelial (SAE) basal cells (BCs). Furthermore, we demonstrated that establishing an air-liquid interface within the bioreactor promoted early differentiation of the SAE BCs [5]. In this article, we orthotopically transplanted these bioengineered tracheas and tracked the contribution of fluorescent airway stem cells.…”