An Improved Double-Chamber Bioreactor for De-epithelialization and Re-epithelialization of Trachea Scaffolds

Lee, Hankyu 1; Marin, Alba 1; Gava, Fabio 2; Karoubi, Golnaz 2; Romero, David 1; Waddell, Tom 2; Amon, Cristina 1

 1.  Advanced Thermal/Fluids Optimization, Modelling and Simulation (ATOMS) Laboratory, Department of Mechanical & Industrial Engineering, University of Toronto; 
 2.  Waddell Research Lab Group, Latner Thoracic Surgery Research Laboratories, University Health Network - MaRS Centre, Toronto Medical Discovery Tower

Tracheal pathologies such as stenosis, malignancy and partial occlusion resulting from traumatic injury affecting more than 50% of the tracheal length usually require long-term dependence on tracheostomies, which carry the risk of repeated infection, excessive bleeding, and thyroid damage. Tracheal transplantation using engineered biological scaffolds are a promising technology to circumvent tracheostomies and its associated complications. 

However, despite recent advancements, successful epithelialization of the tracheal scaffolds – without which cause tracheal stenosis, collapse, fibrosis and infections upon transplantation – remains a challenge for graft success. One obstacle to a successful epithelialization is a lack of an optimized cell seeding protocol with a well-defined set of cell-seeding process variables. This is difficult to achieve as predicting and assessing the cell seeding process experimentally is very time consuming and expensive. 

Hence, this work aims to optimize the re-epithelialization of the tracheal scaffold by defining the critical perfusion cell seeding parameters for an adequate adherence of the tracheal epithelium. To this end, two related objectives are defined: (1) To design, fabricate and validate a bioreactor that is capable of producing flow conditions conducive to the re-epithelialization of the tracheal graft and (2) to predict the spatial distribution of adhered epithelial cells on the tracheal scaffold, using a Computational Fluid Dynamics simulation model (ANSYS Inc., Canonsburg, PA, USA) under different cell seeding parameters. 

As of now, the design, fabrication, and assembly of the bioreactor have been completed. A body-and-lid design was created to allow the tracheal scaffold to remain fully immersed in fluid media during the assembly of the bioreactor and experimental setup, thus helping preserve its viability. The bioreactor includes a custom-made automated, programmable peristaltic pump system to streamline and tightly control fluid flow during re-epithelialization, as well as sensors to monitor the temperature, flow-rate, tracheal rotational velocity and, indirectly, the resulting tracheal lumen wall shear stress in real time. All electronic components communicate with a microcontroller and laptop that logs process control variable data and reads the user inputted process variable settings for a controlled de-epithelialization and re-epithelialization. CFD simulations of cell deposition on the tracheal scaffolds are currently in progress. The combined use of a new platform to perform the tracheal epithelialization and CFD simulation results to optimize the cell seeding protocol are expected to improve experimental outcomes during the de- and re-epithelialization of tracheal scaffolds.