Creation of Live Lung Slices and Analysis of Cell-Matrix Interactions for Regionalized Human Lung Regeneration

Currently, the definitive treatment for end-stage organ failure is orthotropic transplantation, or the transplantation of a whole organ from one patient to another. Due to a critical shortage of viable donor organs, however, the number of unwarranted deaths due to chronic lung disease has increased: an estimated 120,000 deaths per year in the United States alone. Additionally, only about one in five donor lungs are usable for transplantation. In recent years, focus has shifted to regenerative medicine and, specifically, tissue engineering approaches in which cellular sources and scaffolding materials are combined to create functional tissue constructs. Tissue engineering provides a safer alternative to transplantation by minimizing or eliminating most of the aforementioned risks. Thus, the overall objective of this project is to increase the available number of viable lungs by regionally re-conditioning the damaged part(s) of lungs deemed marginally unacceptable for transplant. Re-conditioning includes de-cellularizing damaged or dysfunctional regions of the air space in the lungs Furthermore, some tissue engineering approaches may be incorporated into the patient's own vascular supply for improved blood flow and circulation throughout the implant. The optimal temperature for decellularization and recellularization is 37oC. However, the ideal temperature for vasculature preservation is 4oC. Thus, one significant problem that must be investigated is the effect of varying temperature on the processes required for vasculature maintenance and re-cellularization. In addition, new methods must be established that permit micro-scale analysis of live lung tissue, in particular the vasculature, for this and future studies. Thus, the two goals of the present study were to investigate the attachment of a pulmonary epithelial cell type as a function of temperature as well as to establish a method for generating a thin (<500 μm) live lung slice for future functional studies of the vasculature. Metabolic assays and fluorescent staining were conducted on small airway epithelial cells (SAECs) or cells that are found in lung airways that were grown at different temperatures and different coated plastics. The different coats included gelatin, fibronectin, digested ECM, and a tissue culture plastic control. Results indicated that gelatin was the most effective in cell attachment and that 20oC was the optimal temperature for confluency and cell density for our project's goals. In addition, 12% gelatin perfusion of lung airways was found to be the most effective for the lung slice. Future work includes trying cellular co-cultures and iPS cells for cell attachment and immunological study models on the functional live lung live slice.