Recellularization of decellularized mouse lung airways using negative pressure directed cell delivery

Ahmadipour, Mohammadali (1, 2), Aoki, Fabio Gava (2), Karoubi, Golnaz (1, 2), Waddell, Thomas K. (1, 2)

(1) Institute of Biomaterials and Engineering, University of Toronto

(2) Latner Thoracic Surgery Research Laboratories, Toronto General Hospital Research Institute, University Health Network

For end-stage lung disease, lung transplantation remains the only viable treatment approach. Transplantation, however, is limited due to the availability of transplantable lungs. Generation of bioartificial lungs through the process of decellularization and recellularization is an exciting alternative currently under investigation in the field of tissue engineering. While there has been significant progress in the optimization of decellularization methodologies, optoimal recellularization of acellular lung scaffolds remains a challenge. In this project, we hypothesized that the control of mechanical parameters, for example, using negative pressure, would enhance airway epithelial repopulation. Specifically controllable fluid flow generated via negative pressure may allow direct cell distribution in site-specific areas within proximal and distal regions in the lung airways. we evaluated the effect of negative pressure during cell delivery on cell distribution. Human bronchial epithelial cells (2.5 million cells in a volume of 1.5 ml of cell culture media) were used to seed acellular lungs. Cells were seeded using gravity-based perfusion (control) or via negative pressure (generating a vacuum around the lung by removing air) at rates of 200 ml/ min, 50 ml/min and 12.5 ml/ min. After cell delivery, cells were maintained without movement for 18 hrs and stained using hematoxylin and eosin (H&E). Slides were scanned and analyzed by semi-quantitative subjective scoring and using an image analysis platform (HaloTM). Cell surface coverage was determined using the following formula: Cell surface area/(Cell surface area + scaffold area). A One Way ANOVA with a Dunnett’s post hoc test was used to compare the results and evaluate statistical significance.Our results demonstrate that negative pressure cell seeding results in enhanced overall cell distribution p < 0.05 in for a specific range of fluid flow rates (200 – 50 ml/min) in comparison to the gold standard method of gravity perfusion cell seeding. Moreover, we observed that increasing the fluid flow rate in negative pressure cell seeding protocol increases cell surface coverage.We conclude that negative pressure cell seeding is a superior method of cell delivery in decellularized lung scaffolds in comparison to gravity-based perfusion. Further studies will focus on evaluating the effect of negative pressure cell seeding flow rates on targeted delivery of cells to specific regions of the decellularized scaffold.